Microfluidic synthesis of chiral salen Mn(II) and Co(II) complexes containing lysozyme

Abstract

Efficient microfluidic synthesis of chiral salen Mn(II) and Co(II) complexes containing lysozyme was achieved. The reaction conditions for the delicate protein were controlled precisely with a separation barrier in the microfluidic device. The microfluidic method has the following advantages over the conventional method: a remarkable reduction in the reaction time from 4.5 h to less than 1 s for the synthesis of Mn(II) and Co(II) complexes, a reduction in the reaction temperature from 40 to 23 °C, and no need for an N₂ atmosphere because all the reagents are isolated from the air. A three-fold yield improvement was achieved for Mn(II) and Co(II) complexes containing lysozyme compared with conventional synthesis.

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1. Introduction

Recently, many researchers have focused on synthesizing metal complexes containing proteins, which can be used in drug discovery or fuel cells. For example, Das et al.¹ and Fani et al.² reported that functional complexes exhibited antimycobacterial activities against a Mycobacterium tuberculosis strain. Because these complexes are less toxic to healthy cells, they are promising for drug discovery. In contrast, Nogala and Wittstock³ described the oxidation-reduction potential of laccase. They used a film containing laccase and Fe(CN)₆ as a mediator to build a biologically safe biofuel cell. However, these methods were inefficient, because altering the protein using organic solvents and ligands required a long synthesis time.

Many researchers have used conventional-scale glassware to synthesize complexes containing proteins. However, the reaction time is too long at this scale, and the oxidation of metal compounds by atmospheric oxygen is a major problem. Furthermore, the complex must be separated from the unreacted compounds and organic solvent after the synthesis. For instance, Akitsu's group reported the importance of atmosphere control and separation in their studies on the synthesis of a metal complex containing lysozyme.⁴ Microfluidic devices have been widely used for chemical synthesis.^5,6 Stone's group calculated the diffusion rate in a microfluidic device,⁷ and Atencia and Beebe reviewed microfluidic techniques.⁸ In particular, microscale laminar flow provides efficient and stable synthesis results. Cygan and Amis reported that polydimethylsiloxane (PDMS) is a useful material because of its resistance to water and alcohol.⁹ Therefore, PDMS devices have been used in many practical studies for organic chemical reactions^10–12 and metal ion extraction.¹³ Furthermore, a Cu(II) complex has been separated in a microfluidic PDMS device.¹⁴

Microscale devices have achieved improved reaction speeds and the highest yields.^15–17 Ismagilov's group described the efficient synthesis of metal complexes using a reaction and sample handling with a short diffusion length and on a small scale.¹⁸ We have also synthesized a variety of metal complexes with simple microfluidic devices.^19,20 Vargas-Bernal reported a set of topologies based on microfluidic systems to optimize the design of biosensors for pesticides.²¹

In this study, we compared the synthesis of chiral salen Mn(II) and Co(II) complexes containing lysozyme in conventional glassware and in a PDMS microfluidic device (Fig. 1). Because microfluidic chemical synthesis allows fast reactions, we expected that the reaction time would be reduced at low temperatures and that the enclosed channel walls would suppress the effect of the atmosphere. The synthetic process and results were evaluated with infrared (IR) spectroscopy, scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), ultraviolet-visible (UV-vis) spectroscopy, and mass spectroscopy (MS).

Fig. 1 Comparison of syntheses using the microfluidic device and the conventional method. (a) Laminar flow synthesis using the microfluidic device. (b) Differences in the atmospheres used for the syntheses.

2. Experimental

2.1 Conventional synthesis

As shown in Fig. 2, the Mn(II) and Co(II) complexes containing lysozyme were synthesized in three steps following the method reported by Akitsu and co-workers.⁴ Steps I, II, and III are the synthesis of the ligand, metal complex, and metal complex containing the protein, respectively (Fig. 2). In steps I and II, the product was synthesized under a nitrogen atmosphere. The product was analyzed by IR spectroscopy (FT/IR-6200, JASCO), SEM (SU8240, Hitachi), EDX (Genesis-AP2, EDAX), UV-vis spectroscopy (U-3900, Hitachi), and MS (JMS-T100LC, JEOL).

Fig. 2 Three-step synthesis of metal complexes containing proteins.

2.1.1 Synthesis of ligand (step I). 3,5-Dichlorosalicylaldehyde (40 mmol L⁻¹, reagent A) was added dropwise to a solution of (1R,2R)-(+)-1,2-diphenylethylenediamine (20 mmol L⁻¹, reagent B) dissolved in methanol (50 mL), and stirred at 40 °C for 2 h to give a yellow solution of the ligand (IR (KBr (cm⁻¹)): 1622–1598 (C=N)).

2.1.2 Synthesis of Mn(II) and Co(II) complexes (step II). Co(II) or Mn(II) acetate tetrahydrate (20 mmol L⁻¹, reagent C) was added to the resulting solution to give a red solution of the complex. After stirring for 2.5 h under N₂ gas, the complex was filtered off as a red prismatic precipitate (Fig. 3). The product was analyzed by SEM, EDX, UV-vis spectroscopy, and MS.

Fig. 3 Synthesis of diphenyl complexes.

2.1.3 Synthesis of Mn(II) and Co(II) complexes containing lysozyme (step III). The Mn(II) or Co(II) complex solution (40 μmol L⁻¹, kept constant) was added to the lysozyme solution (0, 36, 72, or 120 μmol L⁻¹, reagent D) (Fig. 2). Each solution was examined by UV-vis spectroscopy. The synthetic conditions are described in Table 1.

Table 1 Synthetic conditions

	Conventional method	Microfluidic device
Reagent concentration (steps I and II), metal complex	40 μmol L^−1	40 μmol L^−1
Reagent concentration (step III), metal complex containing protein	120 μmol L^−1	120 μmol L^−1
Reaction temperature	40 °C	Room temperature
Flow rate	—	3 μL min^−1
Stirring time	4.5 h	—
Atmosphere	N2 gas	Air
Synthesis scale (steps I and II), metal complex	50 cm^3	0.002 cm^3
Synthesis scale (step III), metal complex containing protein	20 cm^3	0.002 cm^3
Ligand analysis method (step I)	IR
Metal complex analysis methods (step II)	SEM, EDX, UV-vis, MS
Metal complex containing protein analysis method (step III)	UV-vis

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2.2 Synthesis with the microfluidic device

2.2.1 Design and fabrication of the microfluidic device. The designs of the simple Y-junction and X-junction devices used in this study are shown in Fig. 4. The width of the channel was approximately 500 μm, and the depth was approximately 100 μm. The device was fabricated by standard soft lithography (Fig. 5). The negative photoresist SU-8 (SU-8 3050, Kayaku MicroChem) was spin-coated on a silicon substrate. The Si substrate coated with SU-8 was patterned by a UV exposure machine (MA/BA6, SUSS MicroTec) and developed with SU-8 developer to produce a SU-8 mold. The PDMS resin and curing agent (SILPOT 184, Dow Corning Toray) were mixed in a 10 : 1 ratio and poured onto the SU-8 mold. After degassing, the mixture was cured at 75 °C for 1 h, and then the cured PDMS was demolded. High-aspect-ratio flexible parallel PDMS walls were obtained. The PDMS was bonded directly on to an O₂ plasma-treated glass substrate.

Fig. 4 Design of microfluidic devices. (a) Y-junction device (steps I and II). (b) X-junction device (step III).

Fig. 5 Fabrication of the microfluidic device.

Reagents and solutions were introduced by syringe (1750CX, Hamilton) using a syringe pump (KDS-100, KD Scientific). The syringes and device were connected via ETFE (ethylene tetrafluoroethylene) tubing.

2.2.2 Synthesis of ligand (step I). Chemicals of the highest commercial grade available (Tokyo Chemical Industry; Wako) were used as received without further purification. The concentration and reagents used were the same as those for the conventional method.

A simple Y-junction channel was used for step I (Fig. 4(a)). Ligand was synthesized with 3,5-dichlorosalicylaldehyde (40 mmol L⁻¹, reagent A, inlet a) and (1R,2R)-(+)-1,2-diphenylethylenediamine (20 mmol L⁻¹, reagent B, inlet b). Step I synthesis was completed in the first channel of the Y-junction microfluidic device (Fig. 6). (IR (KBr (cm⁻¹)): 1622–1598 (C=N)). Flow rate from inlet a and b was 3 μL min⁻¹.

Fig. 6 Synthesis of metal complexes using the Y-junction channel.

2.2.3 Synthesis of Mn(II) and Co(II) complexes (step II). Step II (metal complex synthesis from the ligand and Mn(II) or Co(II) acetate tetrahydrate (20 mmol L⁻¹, reagent C, inlet c)), which both used methanol as the solvent. Step II synthesis was completed in the second channel of the Y-junction microfluidic device. This synthesis was performed directly after step I with no treatment, and the outlet for step I was connected to the ligand inlet for step II. The flow rate from inlet c was 6 μL min⁻¹. Finally, the product was filtered off and analyzed by SEM, EDX, UV-vis spectroscopy, and MS.

2.2.4 Synthesis of Mn(II) and Co(II) complexes containing lysozyme (step III). Mn(II) and Co(II) complexes containing lysozyme were produced from the Mn(II) complex generated in step II and lysozyme (reagent D) generated in step III (Fig. 2). We used the X-junction device in step III. This device has a separation barrier for separating the desired product. The Mn(II) complex (40 μmol L⁻¹) was dissolved in a small amount of methanol, and added to a sodium citrate buffer solution (pH 4.25). Lysozyme (0, 36, 72, or 120 μmol L⁻¹) was dissolved in a buffer solution, and Mn(II) or Co(II) complexes and the lysozyme solution were injected from inlet a and b in Fig. 4(b), respectively. The flow rate of both solutions was 3 μL min⁻¹. The product was evaluated by UV-vis spectroscopy.

3. Results and discussion

A C=N peak (1622–1598 cm⁻¹) was observed in the IR spectrum of the product obtained after step II, which showed that the ligand was synthesized (Fig. 7) by both the conventional and microfluidic device methods. Fig. 8 shows the crystal morphology and chemical characterization of both metal complexes synthesized by conventional and microfluidic methods. The morphology of the crystals formed by both methods were similar in the SEM images. The results, including the central metal and other elements, were verified by EDX. A 5 nm layer of Pt was deposited to prevent electrical charge buildup. Furthermore, the UV-vis spectra (Fig. 9) showed changes in the detected π–π*, n–π*, and charge transfer transitions, which indicated that the Mn(II) and Co(II) complexes were obtained. In addition, the MS data showed peaks for the Mn(II) (MW: 611.0) and Co(II) complexes (MW: 615) (Fig. 10), indicating that the Mn(II) and Co(II) complexes were synthesized in the microfluidic device.

Fig. 7 IR spectra of the complexes (step I, ligand identification). (a) Mn(II) complex. (b) Co(II) complex.

Fig. 8 SEM image and EDX analysis of the complexes (step II, Mn(II) and Co(II) complex identification). (a) Mn(II) complex. (b) Co(II) complex.

Fig. 9 UV-vis spectra of the complexes (step II, Mn(II) and Co(II) complex identification). (a) Mn(II) complex. (b) Co(II) complex.

Fig. 10 MS spectra of the metal complexes (step II, conventional method and synthesis in the microfluidic device). (a) Mn(II) complexes. (b) Co(II) complexes.

The synthesis was performed under an N₂ atmosphere for the conventional method, and under an air atmosphere for the microfluidic device. Zhang and Cloud reported that oxygen could pass through a PDMS device easily.²² However, in this study, the reaction time was too short for the reagents to react with oxygen because they flowed continuously into the microfluidic device. In the conventional method, the reaction mixture was stirred, and the metal acetate was oxidized. Furthermore, PDMS was not affected by alcohol.²³ The quantities that microfluidic devices can handle are small, although this problem can be solved by stacking microfluidic devices. In microfluidic devices, reactions occur rapidly and are isolated from the air. Therefore, syntheses that cannot be achieved by conventional methods, such as simultaneous organic and complex reactions, can be performed.

Fig. 11 shows the UV-vis spectra of the final products obtained by the conventional method and synthesis in the microfluidic device. The peak shifts of n–π* and π–π* (215 nm) show that the Mn(II) and Co(II) complexes containing lysozyme were obtained at each lysozyme concentration. The peak shifts of n–π* and π–π* (215 nm) confirmed that the final products were synthesized.

Fig. 11 UV-vis spectra of the metal complexes containing lysozyme (step III, conventional method and synthesis in the microfluidic device). (a) Mn(II)-lysozyme complexes. (b) Co(II)-lysozyme complexes.

We synthesized the metal complexes and metal complexes containing a protein using the microfluidic device. The product was examined by IR, SEM, EDX, UV-vis spectroscopy, and MS, and the results were compared with those of complexes obtained using the conventional method. In future work, to use the synthesized product in batteries or medicine, it must be purified. We plan to use the method described in Zheng et al.²⁴ to crystallize the metal complexes containing lysozyme and analyze the crystals by X-ray diffraction (XRD). However, retrieving crystals in our current method is difficult; thus, we intend to design a new microfluidic device to allow easy crystal retrieval.

4. Conclusion

Mn(II) and Co(II) complexes containing lysozyme were synthesized by using microfluidic devices. A reaction area-separated microfluidic device formed the Mn(II) complex in less than 1 s at room temperature, and synthesis occurred under an air atmosphere. The microfluidic device is capable of chemical synthesis similar to the conventional method, although the microfluidic device is better suited to handling valuable and dangerous reagents. In future work, we will crystallize metal complexes containing proteins, because we can determine the crystal structure via XRD analysis, and crystallization will be used in fabricating fuel cell electrodes. The amount of product generated by using the microfluidic device is small. We intend to solve this problem by making a stacked device, which should enable practical chemical synthesis.

Acknowledgements

This work was partly supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan through a Grant-in-Aid for Scientific Basic Research (S), No. 23226010. The authors also thank the MEXT Nanotechnology Platform Support Project of Waseda University.

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