An enzyme-immobilization method for integration of biofunctions on a microchip using a water-soluble amphiphilic phospholipid polymer having a reacting group

Abstract

A water-soluble phospholipid polymer having an active ester group in the side chain, poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-p-nitrophenyloxycarbonyl polyethyleneglycol methacrylate (MEONP) (PMBN), was used for the immobilization of an enzyme on a plastic microchip. The MPC polymers with BMA units were adsorbed onto the poly(methyl methacrylate) (PMMA) microchip, and the active ester group in the MEONP unit reacted with the amino groups of the proteolytic enzyme, trypsin. Trypsin was immobilized on the sample reservoir, and catalyzed the hydrolysis of the fluorescently labeled ArgOEt to Arg. The consequent separation of product from the substrate, and their detection, were integrated on the microchip and this meant that all procedures from the enzymatic activity to product detection were completed in less than three minutes.

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Introduction

In the past decade, there has been an explosion of interest in the fields of microfluidic systems.^1–7 Microfabricated fluidic devices are potentially powerful tools for chemical or biological assays. These devices offer a rapid analysis, reduced sample consumption and cost. Specific and efficient biological reactions may significantly benefit with microfluidic devices that offer the means for handling small volume samples (i.e., nL to µL) in confined reaction zones. Various biological assays have been performed in these devices, such as enzymatic reactions^2,3 or immunoassays.^4,5 These reactions were performed using a homogeneous enzyme in a channel,² or immobilized enzymes³ or an antibody⁵ on packed beads, or an encapsulated enzyme in a hydrogel.⁶

Recently, much attention has been devoted to the use of phospholipid polymer materials for modifying the surfaces of biomaterials in order to improve their biocompatibility, namely the ability to inhibit nonspecific protein adsorption or cell adhesion.^8–10 In the field of capillary electrophoresis, various kinds of phospholipids are used as a wall coating for the prevention of protein adsorption.^11,12 Ishihara et al. developed new polymeric materials having the typical phospholipid polar group, 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymer, with various alkyl methacrylates or styrene to change the characteristics, such as solubility, so as to be adapted to the surroundings.^13,14 The MPC polymers are very useful for not only artificial organs but also for medical devices and pharmaceutical applications due to their excellent properties; i.e., resistance to protein adsorption and cell adhesion by adsorption onto the substrate surface.^15,16 To extend the biological applications, the phospholipid polymer for bioconjugation, poly[MPC-co-n-butyl methacrylate (BMA)-co-p-nitrophenyloxycarbonyl polyethyleneglycol methacrylate (MEONP)] (PMBN) (Fig. 1), was designed and synthesized.¹⁷ The MEONP unit has an active ester group in the side chain, which can conjugate with a specific biomolecule via its hydrolysis.

Fig. 1 Chemical structure of PMBN.

Considering that PMBN has the hydrophobic unit, BMA, it was anticipated that the PMBN could be adsorbed on the poly(methyl methacrylate) (PMMA) surface and mediate the conjugation of biomolecules such as enzymes via the active ester units. In this study, we developed a novel enzyme-immobilization method on a PMMA microchip using PMBN for integration of the biofunctions on the microchip.

Experimental

Materials and chemicals

4-Fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and anhydrous ethylenediamine were purchased from Tokyo Kasei (Tokyo, Japan). Trypsin from porcine pancreas and L-Arg were purchased from Sigma-Aldrich (Milwaukee, WI). Arg ethyl ester dihydrochloride was purchased from Wako Pure Chemicals (Osaka, Japan). The PMBNs were synthesized as described in the previous report.¹⁷ Two kinds of PMBN were tested, and the mole fractions of each monomer unit in the PMBN were MPC/BMA/MEONP = 0.4/0.5/0.1 and 0.3/0.6/0.1, described as PMBN40 and PMBN30, respectively. Water was purified by MilliQ apparatus (Millipore, Bedford, MA).

Apparatus

Separations were performed on a microfabricated PMMA chip, i-chip 3 DNA (Hitachi, Tokyo, Japan). The schematic layout is shown in Fig. 2. The microchip has dimensions of 85 mm × 50 mm square with three simple cross channels of 100 µm in width and 30 µm in depth. The distance between the sample reservoir (SR) and the sample waste (SW) was 10 mm, whereas the distance from the buffer reservoir (BR) and the buffer waste (BW) was 44 mm. A small plastic tip was attached to each reservoir and each waste for sample or buffer introduction and removal, respectively.

Fig. 2 Diagram of i-chip3 DNA and schematics of trypsin immobilized on a microchip via PMBN. The area within the dotted line is one cross channel. SR: sample reservoir, SW: sample waste, BR: buffer reservoir, BW: buffer waste.

The microchip electrophoresis system was the same as described in our previous report.⁶ 50 mM Tris–HCl buffer (pH 7.5) was used as the running buffer. The voltage settings were also based on a previously published report.⁶ In all experiments, the potentials of 100, −90, −500 V were applied to SR, BR and SW, respectively, for the 40 s sample introduction, while BW was grounded. This ensured minimal sample introduction bias in the injection cross section. The voltage program for the separation of analytes was 500, 900, 500 V for SR, BR and SW, respectively, and BW was grounded.

After every run, each reservoir was replaced with fresh buffer. A cleaning procedure was performed by flowing the buffer in the channel for 2 min using the same voltage program as that for sample separation.

(Caution! The electrophoresis uses high voltages and special care should be taken when handling the electrophoresis electrodes.)

Derivatization of samples with NBD-F

Samples were derivatized with NBD-F according to the procedure by Imai et al.¹⁸ Derivatized samples were diluted with the running buffer. Before use, all solutions were filtered through a 0.22 µm membrane (Millipore, Bedford, MA) and degassed by ultrasonication.

Trypsin immobilization on the microchip

All channels were filled with 50 mM Tris–HCl (pH 7.5) and the other three reservoirs except for the SR were filled with 50 µL of the buffer to prevent the PMBN solution from permeating into the channels and the other reservoirs. A 10 µL volume of the 0.3% (w/v) PMBN aqueous solution was carefully added to the SR and allowed to stand for 5 min so that the PMBN could adsorb onto the PMMA surface of the SR. After the solution was removed, the SR was dried using air. The PMBN solution was again added to the SR and the same procedure was repeated once more. A 10 µL solution of trypsin (10% (w/v)) in 50 mM Tris–HCl (pH 7.0) containing 20 mM CaCl₂ was carefully added to the SR and allowed to react with the adsorbed PMBN at 4 °C for 2 days. After the unreacted trypsin was removed, the SR was washed and filled with 50 mM Tris–HCl (pH 7.5) before analysis.

On-chip tryptic digestion

Before reaction, the channels were filled with 50 mM Tris–HCl (pH 7.5). Also the three reservoirs except for the SR were filled with 50 µL of the same buffer. A 40 µL volume of the substrate solution containing an internal standard, NBD–ethylenediamine, was added to the SR and voltage was applied immediately.

Results and discussion

The chemical structure of PMBN is shown in Fig. 1. PMBN consists of three components, MPC, BMA, and MEONP units. They were polymerized by a conventional radical polymerization technique in ethanol using 2,2′-azobisisobutyronitrile as the initiator. PMBN is amphiphilic, having both a hydrophilic MPC unit and a hydrophobic BMA unit.¹⁷ In aqueous solution, BMA forms aggregates and adsorbs onto the hydrophobic substrate surface; in this case, the PMMA chip surface. On the other hand, the hydrophilic MPC unit is oriented toward the aqueous solution. MEONP works as an active ester unit to conjugate with specific biomolecules having amino groups. In this study, trypsin, a proteolytic enzyme, was immobilized on the SR surface via the hydrolysis of the active ester units for the fabrication of the biochip which integrates the tryptic reaction of the substrate, separation of products, and detection (Fig. 2).

After PMBN was adsorbed onto the SR surface, the trypsin solution was added to the SR for immobilization. The reaction time for the trypsin immobilization was examined, and it was optimized by measuring the enzymatic activity. The enzymatic activity increased with a reaction time for the trypsin immobilization up to 2 days. For any longer time, the enzymatic activity was not changed. Therefore, 2 day reaction was thought to be enough for the trypsin immobilization on the SR. When trypsin solution was added to the SR without adsorption of PMBN and removed after 2 days, the tryptic activity was not observed, which indicates that the non-covalent adsorption of trypsin on the well surface was negligible.

We used two kinds of PMBN, that is, PMBN40 and PMBN30. Trypsin immobilized via PMBN40 was not stable, and the activity decreased to 20% of the initial activity after 5 analyses. On the other hand, trypsin immobilized via PMBN30 was more stable and the activity was not changed after 15 analyses. Presumably, PMBN30 having a more hydrophobic BMA unit than PMBN40 facilitated its adsorption onto the PMMA surface and contributed to the stability of the trypsin activity.

Fig. 3 illustrates the typical electrophorogram of NBD–ArgOEt and NBD–Arg along with the internal standard, NBD–ethylenediamine, when 1.0 mM NBD–ArgOEt was introduced into the microchip. Immobilized trypsin successfully hydrolyzed NBD–ArgOEt to NBD–Arg, and 0.45 mM NBD–Arg was produced. Furthermore, the three peaks were completely separated in 120 seconds. The migration times were almost the same as those separated on a chip without PMBN and trypsin coating, which indicates that the PMBN and trypsin coating has little influence on electroosmotic mobility.

Fig. 3 Electrophorogram of an on-chip tryptic reaction using NBD–ArgOEt as a substrate. Conditions: Sample: 0.1 mM I.S., 1 mM NBD–ArgOEt. Detection point: 16 mm. Internal standard (I.S.): NBD–ethylenediamine.

This microchip can be used for at least 2 days. On the other hand, a soluble trypsin, which was stored in the same running buffer of 50 mM Tris–HCl (pH 7.5) at 25 °C, almost completely lost its activity within 1 day.¹⁹ These results show the stability of the linkage of trypsin to well surfaces, and that the stability of trypsin was enhanced by immobilization because autolysis of the enzyme was prevented.⁶

Conclusion

The water-soluble amphiphilic phospholipid polymer, PMBN, could be used for the immobilization of an enzyme on a PMMA microchip. The preparation method is very easy, and this method is applicable to a variety of biomolecules with amino groups to react with PMBN. This technique will permit the fabrication of a biochip that contains a discrete area for bioreactions and further expands the scope of microchip analysis.

Acknowledgements

This work was supported by grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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