- Email: [email protected]
Microfluidic Array Liquid Chromatography: A Proof of Principle Study
CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 47, Issue 4, April 2019 Online English edition of the Chinese language journal
Cite this article as: Chinese J. Anal. Chem., 2019, 47(4): 500–507
RESEARCH PAPER
Microfluidic Array Liquid Chromatography: A Proof of Principle Study ZHOU Zhuo-Heng, LIU Ya, ZHANG Bo* College of Chemistry & Chemical Engineering, Xiamen University, Xiamen 361005, China
Abstract: Miniaturization of separation techniques can minimize consumption of solvent and requirement of sample size and leads to high efficiency, high speed, high resolution and high throughput analysis. In this study, a microfluidic chip-based array liquid chromatographic platform enabling multicolumn separation and detection was developed. The platform is based on the combination of glass, poly(methyl methacrylate) (PMMA) and polydimethylsiloxane (PDMS) chips and up to 8 packed chromatographic channels were manufactured on such a microfluidic device. Pressurization experiments showed that the array chip could be reliably used under pressure-driven liquid chromatographic mode. A nanoflow of 300 nL min–1 per column was realized through multi-stage flow splitting. We investigated the feasibility and effectiveness of 8 column array chip and obtained a high efficiency of 80000 plates per meter and a RSD of 1.1% for the retention time. Using a protein digest as the sample, we also explored the potential applicability of the chip in high throughput separation of complex biomolecules. Key Words:
1
Microfluidics; Liquid chromatography; Chip chromatography; Multicolumn array; High throughput
Introduction
Since electrophoresis units were first introduced to microchips by Manz et al[1,2] in 1990s, microfluidic electrophoresis technology achieved a rapid and significant development. Due to the very similar hydrodynamic dimensions of channels in capillaries and in microchips, various modes of capillary electrophoresis (CE) techniques are transformed smoothly into chip-based, showing a widely applied prospect. Compared with electrophoresis, liquid chromatography (LC) features better reproducibility and stability. In addition, due to the multiplex interaction mechanism between mobile phase and stationary phase, LC is capable of handling a broader range of analytes species. Developed for several decades, HPLC has become the most important separation and analytical techniques in drug discovery, environmental protection, food safety and life science researches. However, due to the challenges form column packing, interfacing, sampling and automation
techniques, the development of chip-LC was far more behind that of chip electrophoresis[3]. With regard to the miniaturization of liquid chromatography system, capillary based narrow-bore LC and more recently nano-LC are representing the state of the art, and gradually becoming the working horses toward proteomics analysis. Nano-LC coupled with mass spectrometry is now dominating in large-scale proteomics identification. Compared with capillary, microfluidics chips have a similar dimension, and are expected to satisfy all the technical demands in terms of miniaturization of LC. Meanwhile, micro manufacture techniques, especially one-step molding technology, eliminate the dead volume effectively caused by improper connection. Further, not only the sampling units and the targeted component extraction units can be integrated with separation channels in series, but also the separation channels themselves can be parallel integrated into an array, thus, the whole analysis time is reduced and the analysis efficiency is improved significantly. Predictably, chip-LC has a
________________________ Received 7 December 2018; accepted 20 January 2019 *Corresponding author. Email: [email protected] This work was supported by the National Natural Science Foundation of China (Nos. 21475110, 21535007, J1310024), the Fundamental Research Funds for Central Universities of China (No. 20720160051) and the Xiamen Municipal Science and Technology Plan Project (No. 3502Z20173019). Copyright © 2019, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(19)61154-0
ZHOU Zhuo-Heng et al. / Chinese Journal of Analytical Chemistry, 2019, 47(4): 500–507
considerable potential in the separation of samples in proteomics. For chip-LC system itself, manufacture of the chromatography columns (channels) is with no doubt the core of the whole platform. Originated from capillary LC, it is reasonable for chip-LC to borrow the idea of column making in capillary LC, and at the meantime, several in-situ column making techniques are established depended on the development of micro machining. The earliest stage of chip chromatography is based on open tubular column, and the fabricating procedure is simple and most feasible among others. For example, Jacobson et al[4,5] coated C18 onto the wall of a separation channel inside a glass chip to separate dyes, and obtained a theoretical plate height of 4.5 μm. Open tubular column is easy to be fabricated and regenerated, however, because of the low phase ratio and limited capacity, it often results in overloading, and has not been developed much during the past twenty years. To increase the phase ratio and the surface area of the stationary phase, He et al[6] brought up the idea of collocated monolith support structures (COMOSS). Taking advantage of the micromachining technology, a micro pillar array was fabricated by directly etching to support the stationary phase. Coated by polystyrolsulfon acid, this structure was used in reverse mode electrochromatography, and 35000 plates were achieved in a 4.5-cm separation channel. Nevertheless, this micromachining technology was costly, which limited its further investigation and application Unlike the columns where the stationary phase is formed by surface modification, packed columns obviously have more surface area and column capacity. Various commercial packing particles also facilitate the separation of different species of analytes. A series of work on high performance chip LC/MS system were published since 2005 by Yin et al[7] from Agilent. In this design, a 150-mm separation channel was fabricated by laser ablation on a polyimide chip. The column was packed with 3.5-μm Zorbax 300-SB C18 particles and integrated with a nanoliter valve and a ESI spray. Hyphenated with mass spectrometry, it was successfully applied to the analysis of glycosylated and phosphorylated proteins. Similarly, monoliths were also introduced into chip column. Induced by photons or heat, monomers were polymerized in-situ to form a continuous column bed. By surface functionalizing with 3-(trimethoxysilyl)-propyl methacrylate, Zeng et al[8] bonded PAA gel with β- or γ-cyclodextrin onto a PDMS chip for chiral separation of amino acids. Besides, Throckmorton et al[9] polymerized a 7-cm methacrylate monolith on chip by UV initiation for separation of peptides, and 6 kinds of peptides were baseline separated within 45 s. It is worth noting that by far the vast majority of chip based chromatography only evolve one single separation channel, and have not lived up to the potential of fully integration. For the
flourishing biomedical researches, samples are always
complicated with a large number of species and extremely low volumes, which demands the miniaturized analytical tools with high resolution and high throughput. Thus a chip-based multiplex miniaturized separation platform is expected to be an ideal option. The commercialized 96-channel capillary/chip electrophoresis platform has been successfully applied in the first generation of high throughput DNA sequencing[10], in which channels were usually unpacked and easy to fabricate and regenerate. Relatively, there were few reports about multichannel micro/nano chromatography due to the difficulty of the fabrication of the multi-channel chromatographic columns on a chip. In addition, for pressure-driven liquid chromatography, sealing and compression-bearing capacity are the key to the robustness of the whole system[11]. The commonly used PDMS chips are easy to be designed and manufactured, but are limited by their elasticity and permeability when applied in liquid chromatography[12]. Generally, fused capillary, PDMS, PMMA and glass each has its own merits in micro manufacture and separation platform establishment, and it is vital to combine those materials rationally to effectively set up the micro/nano chromatographic separation system. In this study, by taking the advantages of microfluidic chips in integration, a chip-based column array for high throughput chromatographic separation was developed. The early stage results of multiplex chip design and column array fabrication were reported, and the feasibility and effectiveness of chip-based column array were preliminarily evaluated.
2 2.1
Experimental Apparatus and chemicals
The instruments used here were as follows: JKG-2A lithography machine (Shanghai Optical & Machinery Co., China); Nikon L150 metalloscopy (NIKON, Japan); CK-250L ultrasonic drilling machine (Shanotu JInshida Co., China); Centurion Q50 furnace (NEY, USA); Harvard PHD 2000 syringe pump (Harvard, USA); KQ-50DE ultrasonic cleaner (Ultrasonic Instrument, Kunshan); P230 high pressure pump (Dalian Elite Analytical Instrument, China); Manual pump (Unimicro Technologies, USA); PDC-002 plasma processor (Harrick Plasma, USA); Electrothermal drying oven (Shanghai Yiheng Instrument, China); Ultimate 3000 nano pump (Thermo Fisher, USA); 200 nL manual sampling valve (Valco Instrument, NED); ActiPix D100 multiplex UV on-column detector (Paraytec, UK). Chrome etchant was prepared by dissolving 50 g of ceric ammonium nitrate in 12 mL of HClO4 and diluted into 300 mL with pure water. HF-HNO3-H2O (5:10:85, V/V) was used as glass etchant. Dithiothreitol (DTT), iodoacetamide (IAA), trifluoroacetic acid (TFA), angiotensin, cytochrome C and trypsin were purchased from Sigma Aldrich, USA.
ZHOU Zhuo-Heng et al. / Chinese Journal of Analytical Chemistry, 2019, 47(4): 500–507
Acetonitrile was purchased from Merck, Germany. Ultrapure water (18.2 MΩ cm) was prepared through a Milli-Q system (Millipore Filter Co., USA). SG2506 glass substrate with chromium coating and AZ1805 photoresist were obtained from Changsha Shaoguang Microelectronics Corp., China. PMMA chips were purchased from Shenzhen Richang Co., China. Screwier and sealing rubber o-rings were provided by the department’s workshop. PEEK fittings were purchased from IDEX, USA. Fused silica capillary (50 μm ID, 365 μm OD) was provided by Hebei Yongnian Ruifeng Co. Poly (tetra fluoroethylene) (PTFE) sleeves were purchased from Sigma Aldrich, USA. Sylgard 184 PDMS prepolymer and curing regent were obtained from Dow Corning, USA. 2.2 2.2.1
Methods Chip design and fabrication
Figure 1 illustrates a multiplex chip used here. The main structure was constituted by two glass chips bonding to each other. The front-end capillary was connected to the chip through a PEEK fitting. The outlet of each channel was connected with capillary through a PDMS chip from where the eluent was transferred into the optical detection unit downstream. This glass chip was fabricated by photolithography and wet etching, specifically (Fig.2), it could be divided into several
steps as follows: (1) Mask design: A network of microfluidic channels was designed with a CAD program before it transferred onto a 60 cm × 60 cm thin film by a high resolution laser printer. (2) Exposure: Gently blow the patterned mask with nitrogen to remove the dust, after cleaning, the mask was put on the glass substrate with chromium coating. The mask and the substrate were aligned and put together under the aperture of the lithography machine. The UV beam was generated from the light source inside the lithography machine and shattered onto the substrate through the patterned mask, the exposure time was set to 80 s. (3) Developing: NaOH solution (0.5%) was used as developing liquid. The exposed substrate was soaked in the developing liquid for 7 s before it was washed under ultrapure water for 5 min. It was then dried by nitrogen and heated at 135 °C for 15 min to solidify the undeveloped area. (4) Removing chromium layer: The developed substrate was soaked in chrome etchant for 60–70 s before washed by ultrapure water. (5) First etching: The prepared substrate was put into a plastic beaker with glass etchant, the microchannels were etched in a well-stirred water bath at 45 °C for 1 h. The tapered area was covered by a piece of scotch tape during the first etching process. (6) Second etching: The scotch tape was uncovered and the tapered area was exposed in the etchant for another 15 min. (7) Removing excessive photoresist and the chromium layer: The etched substrate was immersed in acetone while gentle stirred until the surface turn into bright yellow. Then, it was immersed in the chrome etchant for several minutes until the substrate turn
Fig.1 Design of array column chromatography chip (A) and real device (B)
Fig.2 Fabrication workflow of glass chip a. Exposure; b. Development; c. Remove Cr layer; d. Etch channel; e. Remove photoresist and Cr layer; f. High temperature bonding
ZHOU Zhuo-Heng et al. / Chinese Journal of Analytical Chemistry, 2019, 47(4): 500–507
into totally transparent. (8) Drilling: A CK-250L ultrasonic drilling machine was used to drill holes at the inlet and outlets of the channels, and the diameters were 2.0 and 0.5 mm, respectively. (9) Pretreatmentbefore bonding: The substrate and the cover plate were washed with acetone, methanol, and ultrapure water in sequence for 10 min each before they were transferred into the piranha solution (H2SO4-H2O2, 3:1, V/V) to be soaked for 12 h. (10) Bonding: The substrate and the cover plate were washed and dried after pretreatment. After alignment, they were subjected to the programmed furnace for thermal bonding, briefly, the temperature rose to 100 °C in 10 min, and gradually ascended to 580 °C in 30 min and maintained for 2 h, then, slowly cooled down back to room temperature. Two silicon wafers were used to cover the both sides of the chip to ensure a clean surface. 2.2.2
Chip column packing
Approximately 1 mg of 5-μm C18 particles were mixed with 1.5 mL of ACN under ultrasound to form the packing slurry. Before packing with a high pressure HPLC pump, a manual pump was used to pre-pack the tapered area to ensure solid frits been generated at the outlets. High pressure HPLC pump was introduced when the column bed increased to 2 cm. Gentle tapping on the stainless steel tube which contained the slurry was effective to prevent clogging and enhance the packing process. The pump was stopped when the column bed finally reached 2.5 cm (observed under a microscopy) and disconnected to the chip until the pressure fell to 0 psi. Sudden pressure fall should be avoided due to the absence of inlet frits.
2.2.3
Interface design and fabrication
To couple the chip column array to the front-end nano-LC pump and the downstream optical detector, a robust interface was in demand. At the inlet, two 3 cm × 8 cm PMMA chips with 4 threaded holes were used as a clamp to compress a modified PEEK fitting against the glass chip. A 0.8-mm OD rubber o-ring was placed between the fitting and the glass chip to prevent leaking under a high pressure. No leakage was observed under 600 psi. This “sandwich” configuration is illustrated in Fig.3. A total of 8 capillaries were connected to the outlets of the glass chip for transferring the eluent down to the UV detector. PDMS chips were therefore used to mediate the connection. PDMS prepolymer were fully mixed with curing regent at mass ratio of 10:1. The mixture was poured into a petri dish and placed into a vacuum dying chamber to degas, and then subjected to an electrothermal drying oven to cure for 2 h. The prepared PDMS chip was cut into several 0.5 cm × 1.0 cm pieces and each was drilled a hole in the center. The drilled pieces were plasma treated with the glass chip before they were adhered to each other. The holes on the PDMS chips and the glass chip were aligned precisely, eight capillaries with PTFE sleeves were inserted into these holes to endow the fluid connection (Fig.4). 2.2.4
Protein digestion
Approximately 6 mg of cytochrome C was dissolved in 150 mL of NH4HCO3 buffer (40 mM, pH 8), then, 48 μL DTT solution
Fig.3 Sandwich configuration for high pressure entry interface design (A) and real device (B)
Fig.4 Design of exit interface (A) and real device (B)
ZHOU Zhuo-Heng et al. / Chinese Journal of Analytical Chemistry, 2019, 47(4): 500–507
(50 mM) was added. The mixture was water bathed at 56 °C for 45 min. After cooled down to the room temperature, 166 μL of IAA solution (55 mM) was added, and it was then placed in dark for 30 min. Trypsin was added at a protein-to-enzyme ration of 50:1, and the digestion was incubated in water at 37 °C for 18 h. Finally, a small amount of formic acid was introduced to quench the reaction. After desalting and buffering, the digestion was stored in freezer at –20 °C. 2.2.5
Chip liquid chromatography
(1) Separation of alkyl benzenes: 200 nL of (25 nL for each column) mixture of methyl-, ethyl-, propyl, butylbenzne was used as sample. The separation was performed under isocratic condition with 60% ACN water solution as mobile phase. (2) Separation of protein digest: For analysis of trypsin digest of cytochrome C, the sample volume was set at 200 nL (25 nL for each column), with H2O + 0.05% TFA as mobile phase A and ACN+0.05% TFA as mobile phase B. The gradient program was as follows: 0–4 min, 5% B; 4–30 min, 5%–60% B; 30–60 min, 60% B. The flow rate of mobile phase was set at 2.4 μL min–1 (300 nL min–1) and the detection wavelength was 214 nm.
3 3.1
Results and discussion Design and fabrication of chip chromatography column
The chip column array used in this study is illustrated in Fig.1. The chip was divided into 4 functional areas, inlet area, splitter area, packed separation channels and outlet area. Because the pressure-driven separation mode was adopted, the main part of the column array was fabricated with glass chip for better pressure-bearing capacity. Besides, processability should also be comprehensively considered when choosing the material for the interface of both inlet and outlet area. For sampling and liquid introducing, a sandwich structure composited by two PMMA chips was used to compress a PEEK fitting against the glass chip, four screws were used to clamp these three layers tightly, effectively preventing any leakage at the interface. At the outlet area, 8 fused silica capillaries were used to export the eluent for the downstream optical detection. Normally, proportional flow splitting is adopted to obtain as well as control the fluids on a micro/nano scale. For instance, a flow rate of 1 μL min–1 can be generated by splitting on the ratio of 1: 1000 from the parent stream flowing at 1 mL min–1. In this study, a splitting solution illustrated in Fig.1 was used to acquire nanoflow in both 8 channels, namely, the mainstream was divided into two branches, and two into four, four into eight, ultimately the flow rate of every channel was one-eighth of the original. Our preliminary experiments demonstrated that
nanoliter-level flow rate could be stably provided under the condition of precise fabrication, which was further proved by the subsequent chromatographic characterization. 3.2
Packing of chip column array
Similar to the most classic column-based liquid chromatography, the packing of the column is vital to the whole separation process. Our group has accumulated several practical experiences with regard to column packing in capillary chromatography, especially the single particle fritting technology as we reported previously has provided an efficient solution to the fabrication of capillary columns[13–18]. Analogically, preparing well-functioned frits is also involved in the packing of chip column array, namely, effectively containing the chromatographic particles inside the packing bed while enabling the mobile phase to flow through. Based on photolithography and micro machinery, we designed a tapered area to narrow down the inner diameter at the tail end of each column, by which the particles were confined due to the key stone effect[13]. The width of the tapered channel was 20 μm, and the key stone effect worked instantly for those packing particles of 5 μm without any bleeding. The eight channels of the column array were packed in sequence. After the flurry was introduced through the inlet, only one channel was remained open at the tail end while the rest was blocked. The packing pump stopped as soon as the bed reached the required length. At the same time, the tail end of the next channel to be packed was opened while the rest 7 ones remained sealed. This cycle was repeated 8 times until all the channels were packed. In particular, the width of the splitting channel was 300 μm, the width of each column was 100 μm, the length of each column was 25 mm, the width of the tapered end was 20 μm and the length of the end was 5 mm, respectively. The morphology of the cross section of the packed bed was characterized by SEM. As shown in Fig.5, the cross section was U-shape due to the isotropic reaction between hydrofluoric acid and glass. The compactness of the packed bed was consistent radially from the center to the wall. 3.3
Establishment of platform
A 100-μm fused silica capillary with PTFE sleeve was threaded into the inlet hole and tightened by the PEEK fitting, and other of the capillary was connected to the high pressure nanoflow pump. In the downstream column array, 8 identical capillaries (10 cm, 50 μm ID) were fitted into the PDMS outlet interfaces, and a 1-cm wide optical detection window was made just 3 cm from the end of each capillary by ablation of the polyimide coating. As shown in Fig.6, the eight capillaries were bundled and placed in the cartridge of the UV multiplex detector.
ZHOU Zhuo-Heng et al. / Chinese Journal of Analytical Chemistry, 2019, 47(4): 500–507
Fig.5 Mask design of chip chromatography columns (A) and SEM of a packed chip column bed (B)
Fig.6 Array chip chromatography platform: (A) array optical detector, (B) total layout of the array chip chromatography system, and (C) cartridge for the 8 column optical detection windows
As for detection, the ActiPix multiplex UV detector that had the capacity of synchronous detection of 1–8 channels was employed. The UV wave length was fixed at 214 nm for the analysis of alkylbenzenes and peptides mixture. 3.4
peaks, at approximately 38 and 39 min) were also evaluated. The RSDs were calculated to be 3.4% and 2.0%, respectively. The great inter-column reproducibility proved the accuracy of
Application and evaluation
The separation efficiency of the chip columns was evaluated under the condition of isocratic gradient. Four alkylbenzenes were selected as standards and separated with a mobile phase of 60% ACN. As shown in Fig.7, all the alkylbenzenes were separated efficiently with good intercolumn reproducibility. By choosing butylbenzene (the last peak) as benchmark, the average separation efficiency of the 8 columns was calculated and 80000 plates m–1 was obtained. Obviously, this result was satisfactory when the packing particle size was 5 μm. Nevertheless, it should be noted that the dead volume caused by the connection still existed, which would therefore compromise the separation efficiency. Digest of cytochrome C was also used to evaluate the chromatography performance by gradient elution, as shown in Fig.8. A satisfactory separation result and an inter-column reproducibility were also demonstrated. The highest peak in the middle of the chromatogram was selected as a benchmark to calculate the inter-column reproducibility of retention time among the eight columns with RSD of 1.1%. Similarly, the inter-column reproducibility of peak area (the last peak at approximately 39 min) and resolution factor (the last two
Fig.7 Isocratic separation of alkylbenzenes (methyl-, ethyl-, propyl-, butylbenzene, in order of elution) on parallel 8 chip columns
Fig.8 Gradient separation of protein digest on parallel 8 chip columns
ZHOU Zhuo-Heng et al. / Chinese Journal of Analytical Chemistry, 2019, 47(4): 500–507
future, we will focus on multiplex sampling techniques and integration of online extraction unit for the cleaning and enrichment of real biological samples, therefore achieving the high throughput screening analysis of bio or clinical samples.
the fabrication process of the chip column array, and meanwhile suggested the stability of the packing method, which ensured the synchronism of separation between each column. It was worth noting that the chip column array assembly consisted of three types of chip units (glass chip, PMMA chip, PDMS chip) and capillaries. Practically, the preparation of each unit needed to be controlled separately, and the assembling of the device was relatively simple. The assembly used here remained steady after one-month test and operation without any clogging and leakage. It should be pointed out that the eight columns inside the chip shared one sample valve in this study, and in this case the same sample was split into eight columns and separated under the same conditions. By this way, the feasibility and effectiveness of the configuration of the chip column array were verified. Besides, we will further investigate its potential of multi samples analysis and even two-dimensional separation in the future work.
[2]
4
[8]
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Conclusions
Zeng H L, Li H F, Lin J M. Anal. Chim. Acta, 2005, 551(1): 1–8
In this study, a multi-channel chromatography chip based on microfluidics technology was developed. On the basis of particle packed column bed, the upstream pressure bearing interface and the downstream capillary connection unit were designed and fabricated, and a multiplex chromatographic separation platform for the high throughput analysis of biosamples was preliminarily established. The results indicated that high separation efficiency and great reproducibility were realized on a column array chip. The fabrication process based on MEMS techniques was expected to be applied in the preparation of large-scale chromatography column array. Featuring in paralleled multiple separation channel, this chip will hopefully play an important role in high throughput analysis. The multiplex chromatographic separation platform based on the chip column array can be potentially coupled with different detectors and integrated with multiple analysis units, which is significant to improve the analytical throughput and resolution of proteomics. Furthermore, this work has only demonstrated the feasibility of on-chip column packing and synchronous separation. In the
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[10] Kircher M, Kelso J. Bioessays, 2010, 32(6): 524–536 [11] Hai Q F, Li J, Wang E K. Chinese J. Anal.Chem., 2018, 46(6): 814‒825 [12] Nge P N, Rogers C I, Woolley A T. Chem. Rev., 2013, 113(4): 2550–2583 [13] Zhang B, Liu Q, Yang L J, Wang Q Q. J. Chromatogr. A, 2013, 1272(1): 136–140 [14] Xiao Z L, Wang L, Liu Y, Wang Q Q, Zhang B. J. Chromatogr. A, 2014, 1325(1): 109–114 [15] Liu Q, Yang L J, Wang Q Q, Zhang B. J. Chromatogr. A, 2014, 1349(7): 90–95 [16] Liu Q, Wang L, Zhou Z H, Wang Q Q, Yan L J, Zhang B. Electrophoresis, 2014, 35(6): 836–839 [17] Han J, Ye L Q, Xu L J, Zhou Z H, Gao F, Xiao Z L, Wang Q Q, Zhang B. Anal. Chim. Acta, 2014, 852(12): 267–273 [18] Yang L J, Xu L J, Guo R, Gao T Y, Guo H X, Yu Y, Lv J J, Wang Q Q, Zhang B. Anal. Chim. Acta, 2018, 1033(11): 205–212
Cite this article as: Chinese J. Anal. Chem., 2019, 47(4): 500–507
RESEARCH PAPER
Microfluidic Array Liquid Chromatography: A Proof of Principle Study ZHOU Zhuo-Heng, LIU Ya, ZHANG Bo* College of Chemistry & Chemical Engineering, Xiamen University, Xiamen 361005, China
Abstract: Miniaturization of separation techniques can minimize consumption of solvent and requirement of sample size and leads to high efficiency, high speed, high resolution and high throughput analysis. In this study, a microfluidic chip-based array liquid chromatographic platform enabling multicolumn separation and detection was developed. The platform is based on the combination of glass, poly(methyl methacrylate) (PMMA) and polydimethylsiloxane (PDMS) chips and up to 8 packed chromatographic channels were manufactured on such a microfluidic device. Pressurization experiments showed that the array chip could be reliably used under pressure-driven liquid chromatographic mode. A nanoflow of 300 nL min–1 per column was realized through multi-stage flow splitting. We investigated the feasibility and effectiveness of 8 column array chip and obtained a high efficiency of 80000 plates per meter and a RSD of 1.1% for the retention time. Using a protein digest as the sample, we also explored the potential applicability of the chip in high throughput separation of complex biomolecules. Key Words:
1
Microfluidics; Liquid chromatography; Chip chromatography; Multicolumn array; High throughput
Introduction
Since electrophoresis units were first introduced to microchips by Manz et al[1,2] in 1990s, microfluidic electrophoresis technology achieved a rapid and significant development. Due to the very similar hydrodynamic dimensions of channels in capillaries and in microchips, various modes of capillary electrophoresis (CE) techniques are transformed smoothly into chip-based, showing a widely applied prospect. Compared with electrophoresis, liquid chromatography (LC) features better reproducibility and stability. In addition, due to the multiplex interaction mechanism between mobile phase and stationary phase, LC is capable of handling a broader range of analytes species. Developed for several decades, HPLC has become the most important separation and analytical techniques in drug discovery, environmental protection, food safety and life science researches. However, due to the challenges form column packing, interfacing, sampling and automation
techniques, the development of chip-LC was far more behind that of chip electrophoresis[3]. With regard to the miniaturization of liquid chromatography system, capillary based narrow-bore LC and more recently nano-LC are representing the state of the art, and gradually becoming the working horses toward proteomics analysis. Nano-LC coupled with mass spectrometry is now dominating in large-scale proteomics identification. Compared with capillary, microfluidics chips have a similar dimension, and are expected to satisfy all the technical demands in terms of miniaturization of LC. Meanwhile, micro manufacture techniques, especially one-step molding technology, eliminate the dead volume effectively caused by improper connection. Further, not only the sampling units and the targeted component extraction units can be integrated with separation channels in series, but also the separation channels themselves can be parallel integrated into an array, thus, the whole analysis time is reduced and the analysis efficiency is improved significantly. Predictably, chip-LC has a
________________________ Received 7 December 2018; accepted 20 January 2019 *Corresponding author. Email: [email protected] This work was supported by the National Natural Science Foundation of China (Nos. 21475110, 21535007, J1310024), the Fundamental Research Funds for Central Universities of China (No. 20720160051) and the Xiamen Municipal Science and Technology Plan Project (No. 3502Z20173019). Copyright © 2019, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(19)61154-0
ZHOU Zhuo-Heng et al. / Chinese Journal of Analytical Chemistry, 2019, 47(4): 500–507
considerable potential in the separation of samples in proteomics. For chip-LC system itself, manufacture of the chromatography columns (channels) is with no doubt the core of the whole platform. Originated from capillary LC, it is reasonable for chip-LC to borrow the idea of column making in capillary LC, and at the meantime, several in-situ column making techniques are established depended on the development of micro machining. The earliest stage of chip chromatography is based on open tubular column, and the fabricating procedure is simple and most feasible among others. For example, Jacobson et al[4,5] coated C18 onto the wall of a separation channel inside a glass chip to separate dyes, and obtained a theoretical plate height of 4.5 μm. Open tubular column is easy to be fabricated and regenerated, however, because of the low phase ratio and limited capacity, it often results in overloading, and has not been developed much during the past twenty years. To increase the phase ratio and the surface area of the stationary phase, He et al[6] brought up the idea of collocated monolith support structures (COMOSS). Taking advantage of the micromachining technology, a micro pillar array was fabricated by directly etching to support the stationary phase. Coated by polystyrolsulfon acid, this structure was used in reverse mode electrochromatography, and 35000 plates were achieved in a 4.5-cm separation channel. Nevertheless, this micromachining technology was costly, which limited its further investigation and application Unlike the columns where the stationary phase is formed by surface modification, packed columns obviously have more surface area and column capacity. Various commercial packing particles also facilitate the separation of different species of analytes. A series of work on high performance chip LC/MS system were published since 2005 by Yin et al[7] from Agilent. In this design, a 150-mm separation channel was fabricated by laser ablation on a polyimide chip. The column was packed with 3.5-μm Zorbax 300-SB C18 particles and integrated with a nanoliter valve and a ESI spray. Hyphenated with mass spectrometry, it was successfully applied to the analysis of glycosylated and phosphorylated proteins. Similarly, monoliths were also introduced into chip column. Induced by photons or heat, monomers were polymerized in-situ to form a continuous column bed. By surface functionalizing with 3-(trimethoxysilyl)-propyl methacrylate, Zeng et al[8] bonded PAA gel with β- or γ-cyclodextrin onto a PDMS chip for chiral separation of amino acids. Besides, Throckmorton et al[9] polymerized a 7-cm methacrylate monolith on chip by UV initiation for separation of peptides, and 6 kinds of peptides were baseline separated within 45 s. It is worth noting that by far the vast majority of chip based chromatography only evolve one single separation channel, and have not lived up to the potential of fully integration. For the
flourishing biomedical researches, samples are always
complicated with a large number of species and extremely low volumes, which demands the miniaturized analytical tools with high resolution and high throughput. Thus a chip-based multiplex miniaturized separation platform is expected to be an ideal option. The commercialized 96-channel capillary/chip electrophoresis platform has been successfully applied in the first generation of high throughput DNA sequencing[10], in which channels were usually unpacked and easy to fabricate and regenerate. Relatively, there were few reports about multichannel micro/nano chromatography due to the difficulty of the fabrication of the multi-channel chromatographic columns on a chip. In addition, for pressure-driven liquid chromatography, sealing and compression-bearing capacity are the key to the robustness of the whole system[11]. The commonly used PDMS chips are easy to be designed and manufactured, but are limited by their elasticity and permeability when applied in liquid chromatography[12]. Generally, fused capillary, PDMS, PMMA and glass each has its own merits in micro manufacture and separation platform establishment, and it is vital to combine those materials rationally to effectively set up the micro/nano chromatographic separation system. In this study, by taking the advantages of microfluidic chips in integration, a chip-based column array for high throughput chromatographic separation was developed. The early stage results of multiplex chip design and column array fabrication were reported, and the feasibility and effectiveness of chip-based column array were preliminarily evaluated.
2 2.1
Experimental Apparatus and chemicals
The instruments used here were as follows: JKG-2A lithography machine (Shanghai Optical & Machinery Co., China); Nikon L150 metalloscopy (NIKON, Japan); CK-250L ultrasonic drilling machine (Shanotu JInshida Co., China); Centurion Q50 furnace (NEY, USA); Harvard PHD 2000 syringe pump (Harvard, USA); KQ-50DE ultrasonic cleaner (Ultrasonic Instrument, Kunshan); P230 high pressure pump (Dalian Elite Analytical Instrument, China); Manual pump (Unimicro Technologies, USA); PDC-002 plasma processor (Harrick Plasma, USA); Electrothermal drying oven (Shanghai Yiheng Instrument, China); Ultimate 3000 nano pump (Thermo Fisher, USA); 200 nL manual sampling valve (Valco Instrument, NED); ActiPix D100 multiplex UV on-column detector (Paraytec, UK). Chrome etchant was prepared by dissolving 50 g of ceric ammonium nitrate in 12 mL of HClO4 and diluted into 300 mL with pure water. HF-HNO3-H2O (5:10:85, V/V) was used as glass etchant. Dithiothreitol (DTT), iodoacetamide (IAA), trifluoroacetic acid (TFA), angiotensin, cytochrome C and trypsin were purchased from Sigma Aldrich, USA.
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Acetonitrile was purchased from Merck, Germany. Ultrapure water (18.2 MΩ cm) was prepared through a Milli-Q system (Millipore Filter Co., USA). SG2506 glass substrate with chromium coating and AZ1805 photoresist were obtained from Changsha Shaoguang Microelectronics Corp., China. PMMA chips were purchased from Shenzhen Richang Co., China. Screwier and sealing rubber o-rings were provided by the department’s workshop. PEEK fittings were purchased from IDEX, USA. Fused silica capillary (50 μm ID, 365 μm OD) was provided by Hebei Yongnian Ruifeng Co. Poly (tetra fluoroethylene) (PTFE) sleeves were purchased from Sigma Aldrich, USA. Sylgard 184 PDMS prepolymer and curing regent were obtained from Dow Corning, USA. 2.2 2.2.1
Methods Chip design and fabrication
Figure 1 illustrates a multiplex chip used here. The main structure was constituted by two glass chips bonding to each other. The front-end capillary was connected to the chip through a PEEK fitting. The outlet of each channel was connected with capillary through a PDMS chip from where the eluent was transferred into the optical detection unit downstream. This glass chip was fabricated by photolithography and wet etching, specifically (Fig.2), it could be divided into several
steps as follows: (1) Mask design: A network of microfluidic channels was designed with a CAD program before it transferred onto a 60 cm × 60 cm thin film by a high resolution laser printer. (2) Exposure: Gently blow the patterned mask with nitrogen to remove the dust, after cleaning, the mask was put on the glass substrate with chromium coating. The mask and the substrate were aligned and put together under the aperture of the lithography machine. The UV beam was generated from the light source inside the lithography machine and shattered onto the substrate through the patterned mask, the exposure time was set to 80 s. (3) Developing: NaOH solution (0.5%) was used as developing liquid. The exposed substrate was soaked in the developing liquid for 7 s before it was washed under ultrapure water for 5 min. It was then dried by nitrogen and heated at 135 °C for 15 min to solidify the undeveloped area. (4) Removing chromium layer: The developed substrate was soaked in chrome etchant for 60–70 s before washed by ultrapure water. (5) First etching: The prepared substrate was put into a plastic beaker with glass etchant, the microchannels were etched in a well-stirred water bath at 45 °C for 1 h. The tapered area was covered by a piece of scotch tape during the first etching process. (6) Second etching: The scotch tape was uncovered and the tapered area was exposed in the etchant for another 15 min. (7) Removing excessive photoresist and the chromium layer: The etched substrate was immersed in acetone while gentle stirred until the surface turn into bright yellow. Then, it was immersed in the chrome etchant for several minutes until the substrate turn
Fig.1 Design of array column chromatography chip (A) and real device (B)
Fig.2 Fabrication workflow of glass chip a. Exposure; b. Development; c. Remove Cr layer; d. Etch channel; e. Remove photoresist and Cr layer; f. High temperature bonding
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into totally transparent. (8) Drilling: A CK-250L ultrasonic drilling machine was used to drill holes at the inlet and outlets of the channels, and the diameters were 2.0 and 0.5 mm, respectively. (9) Pretreatmentbefore bonding: The substrate and the cover plate were washed with acetone, methanol, and ultrapure water in sequence for 10 min each before they were transferred into the piranha solution (H2SO4-H2O2, 3:1, V/V) to be soaked for 12 h. (10) Bonding: The substrate and the cover plate were washed and dried after pretreatment. After alignment, they were subjected to the programmed furnace for thermal bonding, briefly, the temperature rose to 100 °C in 10 min, and gradually ascended to 580 °C in 30 min and maintained for 2 h, then, slowly cooled down back to room temperature. Two silicon wafers were used to cover the both sides of the chip to ensure a clean surface. 2.2.2
Chip column packing
Approximately 1 mg of 5-μm C18 particles were mixed with 1.5 mL of ACN under ultrasound to form the packing slurry. Before packing with a high pressure HPLC pump, a manual pump was used to pre-pack the tapered area to ensure solid frits been generated at the outlets. High pressure HPLC pump was introduced when the column bed increased to 2 cm. Gentle tapping on the stainless steel tube which contained the slurry was effective to prevent clogging and enhance the packing process. The pump was stopped when the column bed finally reached 2.5 cm (observed under a microscopy) and disconnected to the chip until the pressure fell to 0 psi. Sudden pressure fall should be avoided due to the absence of inlet frits.
2.2.3
Interface design and fabrication
To couple the chip column array to the front-end nano-LC pump and the downstream optical detector, a robust interface was in demand. At the inlet, two 3 cm × 8 cm PMMA chips with 4 threaded holes were used as a clamp to compress a modified PEEK fitting against the glass chip. A 0.8-mm OD rubber o-ring was placed between the fitting and the glass chip to prevent leaking under a high pressure. No leakage was observed under 600 psi. This “sandwich” configuration is illustrated in Fig.3. A total of 8 capillaries were connected to the outlets of the glass chip for transferring the eluent down to the UV detector. PDMS chips were therefore used to mediate the connection. PDMS prepolymer were fully mixed with curing regent at mass ratio of 10:1. The mixture was poured into a petri dish and placed into a vacuum dying chamber to degas, and then subjected to an electrothermal drying oven to cure for 2 h. The prepared PDMS chip was cut into several 0.5 cm × 1.0 cm pieces and each was drilled a hole in the center. The drilled pieces were plasma treated with the glass chip before they were adhered to each other. The holes on the PDMS chips and the glass chip were aligned precisely, eight capillaries with PTFE sleeves were inserted into these holes to endow the fluid connection (Fig.4). 2.2.4
Protein digestion
Approximately 6 mg of cytochrome C was dissolved in 150 mL of NH4HCO3 buffer (40 mM, pH 8), then, 48 μL DTT solution
Fig.3 Sandwich configuration for high pressure entry interface design (A) and real device (B)
Fig.4 Design of exit interface (A) and real device (B)
ZHOU Zhuo-Heng et al. / Chinese Journal of Analytical Chemistry, 2019, 47(4): 500–507
(50 mM) was added. The mixture was water bathed at 56 °C for 45 min. After cooled down to the room temperature, 166 μL of IAA solution (55 mM) was added, and it was then placed in dark for 30 min. Trypsin was added at a protein-to-enzyme ration of 50:1, and the digestion was incubated in water at 37 °C for 18 h. Finally, a small amount of formic acid was introduced to quench the reaction. After desalting and buffering, the digestion was stored in freezer at –20 °C. 2.2.5
Chip liquid chromatography
(1) Separation of alkyl benzenes: 200 nL of (25 nL for each column) mixture of methyl-, ethyl-, propyl, butylbenzne was used as sample. The separation was performed under isocratic condition with 60% ACN water solution as mobile phase. (2) Separation of protein digest: For analysis of trypsin digest of cytochrome C, the sample volume was set at 200 nL (25 nL for each column), with H2O + 0.05% TFA as mobile phase A and ACN+0.05% TFA as mobile phase B. The gradient program was as follows: 0–4 min, 5% B; 4–30 min, 5%–60% B; 30–60 min, 60% B. The flow rate of mobile phase was set at 2.4 μL min–1 (300 nL min–1) and the detection wavelength was 214 nm.
3 3.1
Results and discussion Design and fabrication of chip chromatography column
The chip column array used in this study is illustrated in Fig.1. The chip was divided into 4 functional areas, inlet area, splitter area, packed separation channels and outlet area. Because the pressure-driven separation mode was adopted, the main part of the column array was fabricated with glass chip for better pressure-bearing capacity. Besides, processability should also be comprehensively considered when choosing the material for the interface of both inlet and outlet area. For sampling and liquid introducing, a sandwich structure composited by two PMMA chips was used to compress a PEEK fitting against the glass chip, four screws were used to clamp these three layers tightly, effectively preventing any leakage at the interface. At the outlet area, 8 fused silica capillaries were used to export the eluent for the downstream optical detection. Normally, proportional flow splitting is adopted to obtain as well as control the fluids on a micro/nano scale. For instance, a flow rate of 1 μL min–1 can be generated by splitting on the ratio of 1: 1000 from the parent stream flowing at 1 mL min–1. In this study, a splitting solution illustrated in Fig.1 was used to acquire nanoflow in both 8 channels, namely, the mainstream was divided into two branches, and two into four, four into eight, ultimately the flow rate of every channel was one-eighth of the original. Our preliminary experiments demonstrated that
nanoliter-level flow rate could be stably provided under the condition of precise fabrication, which was further proved by the subsequent chromatographic characterization. 3.2
Packing of chip column array
Similar to the most classic column-based liquid chromatography, the packing of the column is vital to the whole separation process. Our group has accumulated several practical experiences with regard to column packing in capillary chromatography, especially the single particle fritting technology as we reported previously has provided an efficient solution to the fabrication of capillary columns[13–18]. Analogically, preparing well-functioned frits is also involved in the packing of chip column array, namely, effectively containing the chromatographic particles inside the packing bed while enabling the mobile phase to flow through. Based on photolithography and micro machinery, we designed a tapered area to narrow down the inner diameter at the tail end of each column, by which the particles were confined due to the key stone effect[13]. The width of the tapered channel was 20 μm, and the key stone effect worked instantly for those packing particles of 5 μm without any bleeding. The eight channels of the column array were packed in sequence. After the flurry was introduced through the inlet, only one channel was remained open at the tail end while the rest was blocked. The packing pump stopped as soon as the bed reached the required length. At the same time, the tail end of the next channel to be packed was opened while the rest 7 ones remained sealed. This cycle was repeated 8 times until all the channels were packed. In particular, the width of the splitting channel was 300 μm, the width of each column was 100 μm, the length of each column was 25 mm, the width of the tapered end was 20 μm and the length of the end was 5 mm, respectively. The morphology of the cross section of the packed bed was characterized by SEM. As shown in Fig.5, the cross section was U-shape due to the isotropic reaction between hydrofluoric acid and glass. The compactness of the packed bed was consistent radially from the center to the wall. 3.3
Establishment of platform
A 100-μm fused silica capillary with PTFE sleeve was threaded into the inlet hole and tightened by the PEEK fitting, and other of the capillary was connected to the high pressure nanoflow pump. In the downstream column array, 8 identical capillaries (10 cm, 50 μm ID) were fitted into the PDMS outlet interfaces, and a 1-cm wide optical detection window was made just 3 cm from the end of each capillary by ablation of the polyimide coating. As shown in Fig.6, the eight capillaries were bundled and placed in the cartridge of the UV multiplex detector.
ZHOU Zhuo-Heng et al. / Chinese Journal of Analytical Chemistry, 2019, 47(4): 500–507
Fig.5 Mask design of chip chromatography columns (A) and SEM of a packed chip column bed (B)
Fig.6 Array chip chromatography platform: (A) array optical detector, (B) total layout of the array chip chromatography system, and (C) cartridge for the 8 column optical detection windows
As for detection, the ActiPix multiplex UV detector that had the capacity of synchronous detection of 1–8 channels was employed. The UV wave length was fixed at 214 nm for the analysis of alkylbenzenes and peptides mixture. 3.4
peaks, at approximately 38 and 39 min) were also evaluated. The RSDs were calculated to be 3.4% and 2.0%, respectively. The great inter-column reproducibility proved the accuracy of
Application and evaluation
The separation efficiency of the chip columns was evaluated under the condition of isocratic gradient. Four alkylbenzenes were selected as standards and separated with a mobile phase of 60% ACN. As shown in Fig.7, all the alkylbenzenes were separated efficiently with good intercolumn reproducibility. By choosing butylbenzene (the last peak) as benchmark, the average separation efficiency of the 8 columns was calculated and 80000 plates m–1 was obtained. Obviously, this result was satisfactory when the packing particle size was 5 μm. Nevertheless, it should be noted that the dead volume caused by the connection still existed, which would therefore compromise the separation efficiency. Digest of cytochrome C was also used to evaluate the chromatography performance by gradient elution, as shown in Fig.8. A satisfactory separation result and an inter-column reproducibility were also demonstrated. The highest peak in the middle of the chromatogram was selected as a benchmark to calculate the inter-column reproducibility of retention time among the eight columns with RSD of 1.1%. Similarly, the inter-column reproducibility of peak area (the last peak at approximately 39 min) and resolution factor (the last two
Fig.7 Isocratic separation of alkylbenzenes (methyl-, ethyl-, propyl-, butylbenzene, in order of elution) on parallel 8 chip columns
Fig.8 Gradient separation of protein digest on parallel 8 chip columns
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future, we will focus on multiplex sampling techniques and integration of online extraction unit for the cleaning and enrichment of real biological samples, therefore achieving the high throughput screening analysis of bio or clinical samples.
the fabrication process of the chip column array, and meanwhile suggested the stability of the packing method, which ensured the synchronism of separation between each column. It was worth noting that the chip column array assembly consisted of three types of chip units (glass chip, PMMA chip, PDMS chip) and capillaries. Practically, the preparation of each unit needed to be controlled separately, and the assembling of the device was relatively simple. The assembly used here remained steady after one-month test and operation without any clogging and leakage. It should be pointed out that the eight columns inside the chip shared one sample valve in this study, and in this case the same sample was split into eight columns and separated under the same conditions. By this way, the feasibility and effectiveness of the configuration of the chip column array were verified. Besides, we will further investigate its potential of multi samples analysis and even two-dimensional separation in the future work.
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In this study, a multi-channel chromatography chip based on microfluidics technology was developed. On the basis of particle packed column bed, the upstream pressure bearing interface and the downstream capillary connection unit were designed and fabricated, and a multiplex chromatographic separation platform for the high throughput analysis of biosamples was preliminarily established. The results indicated that high separation efficiency and great reproducibility were realized on a column array chip. The fabrication process based on MEMS techniques was expected to be applied in the preparation of large-scale chromatography column array. Featuring in paralleled multiple separation channel, this chip will hopefully play an important role in high throughput analysis. The multiplex chromatographic separation platform based on the chip column array can be potentially coupled with different detectors and integrated with multiple analysis units, which is significant to improve the analytical throughput and resolution of proteomics. Furthermore, this work has only demonstrated the feasibility of on-chip column packing and synchronous separation. In the
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