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On-chip lipid extraction using super absorbent polymers for mass spectrometry Geul Bang, Young Hwan Kim, Junghyo Yoon, Yeong Jun Yu, Seok Chung, and Jeong Ah Kim Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03547 • Publication Date (Web): 22 Nov 2017 Downloaded from http://pubs.acs.org on November 22, 2017
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Analytical Chemistry
On-chip lipid extraction using super absorbent polymers for mass spectrometry Geul Bang1,†, Young Hwan Kim1,2,3,†, Junghyo Yoon4, Yeong Jun Yu1,5, Seok Chung4, Jeong Ah Kim1,2,* 1
Biomedical Omics Group, Korea Basic Science Institute, Chungbuk 28119, Republic of Korea. Department of Bio-Analytical Science, University of Science and Technology, Daejeon 34113, Republic of Korea. 3 Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon 34134, Republic of Korea. 4 Department of Mechanical Engineering, Korea University, Seoul 02841, Republic of Korea. 5 Program in Micro/Nano System, Korea University, Seoul 02841, Republic of Korea. * Corresponding author: E-mail:
[email protected]. Fax: +82 43 240 5158 2
ABSTRACT: Pre-treatment of samples is the one of most important steps in analytical methods for efficient and accurate results. Typically, an extraction method used for lipid analysis with mass spectrometry is accompanied by complex liquid-liquid extraction. We have devised a simple, rapid, and efficient lipid extraction method using super absorbent polymers (SAPs) and developed a highthroughput lipid extraction platform based on a microfluidic system. Since SAPs can rapidly absorb an aqueous solution from a raw sample and convert it into the gel, the lipid extraction process can be remarkably simplified. The hydrophobic lipid components were captured into the fibrous SAP gel and then solubilized and eluted directly into the organic solvent without significant interference by this polymer. The small-scale lipid extraction process minimizes the liquid handling and unnecessary centrifugation steps, thereby enabling the implementation of a SAP-integrated microfluidic lipid extraction platform. The SAP method successfully induced reproducible extraction and high recovery rates (95-100%) compared to the conventional Folch method in several lipid classes. We also demonstrated the feasibility of the SAP method for the analysis of lipids in complex biological samples, such as the brain and liver, as well as E. coli. This small-scale SAP method and its microfluidic platform will open up new possibilities in high-throughput lipidomic research for diagnosing diseases because this new technique saves time, labor, and cost.
Lipids are one of the major components of biological cell membranes, and they participate in many biochemical functions and metabolic processes, such as intercellular signaling, secretion, and energy storage.1-3 Recent studies have proven that lipids have key roles in biological mechanisms and they are involved in the lipid-related diseases, such as diabetes, Alzheimer’s disease, atherosclerosis, and cancer.4-6 These findings emphasize the importance of research in the field of bioanalytical chemistry that involves comprehensive and quantitative studies of lipids. For this reason, it is essential that a simple and rapid treatment technique for lipid analysis be developed. Despite the highly-sophisticated analytical process, the successful analysis is determined by the efficient extraction and separation of complex lipid mixtures derived from various biological origins, such as cells, tissues, and body fluids. However, it is very hard to simultaneously analyze all lipid classes with a high recovery rate because such analysis is complicated by the diverse subclasses of lipids, depending on their polar nature and structure of molecular backbone.7-9 Another difficulty in analyzing lipids is that their concentrations vary over a wide range in various samples. As a result, the less abundant species can easily get lost during inefficient sample pre-treatment.10-12 The traditional lipid extraction methods, such as the Folch method and the Bligh-Dyer method, are liquid-liquid extraction
(LLE) methods that involve several consecutive steps.13-15 In general, the LLE method is unsuitable for small quantities of samples because it requires several hours for the extraction and generally has a low recovery rate. These are serous considerations because the removal of water is a prerequisite when treating aqueous samples, such as plasma, urine, and other body fluids. Some time ago, a solid-phase extraction (SPE) method that is referred to as the “quick, easy, cheap, effective, rugged, and safe” method (QuEChERS method) was introduced for various pre-treatments for pesticides, food samples and environmental samples and lipids.16,17 The QuEChERS method is easy and simple to use, and it reduces the extraction cleanup time (< 30 min).18 However, there is still a need to improve this method to obtain reproducible data for lipid analyses because the current method requires difficult extraction/partitioning of the aqueous and organic phases from the solid phase. The accompanying sludge-like solids make it difficult to separate the aqueous phase from the organic phase, and significant losses of the extracted material occur. Ways to overcome these issues and improve the extraction efficiency are to minimize the liquid transfer and reduce the dead volume by using a microchip platform. A miniaturized device comprised of small structures, such as holes and channels,
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can reduce the steps of experimental process and efficiently reduce the volume of the sample that is required. In the field of analytical chemistry, the numerous devices integrated with the sample pre-treatment process have been suggested.19-21 However, there have been relatively few reports of microfluidic devices associated with sample pre-treatment for lipidomic analyses.22, 23 In this article, we describe a miniaturized, solid-phase extraction (SPE) method for lipid samples. The use of super absorbent polymers (SAPs) to absorb aqueous solutions provides a remarkable reduction in the conventionally-required sophisticated steps, such as centrifugation and the transfer of liquids. We have first demonstrated whether this polymer can be utilized in the pre-treatment of samples for efficient analyses of lipids through the following investigations. The lipids are captured effectively within the SAP gel and eluted into the organic solvent. This process does not require long incubation or centrifugation, resulting in a remarkable shortening of the extraction process (5~10 min). One of the most significant advantages of this novel extraction method is the easy integration with a microfluidic system using only two simple reservoirs. Herein, we analyzed the samples, such as a mixture of lipids, tissue extracts, bacterial cells, and plasma to demonstrate that the use of SAPs and a SAPintegrated chip are useful for the extraction of lipids. We evaluated the extraction efficiency and the reproducibility of this method by using quantitative mass spectrometry (MS) analyses to compare our results with those of the modified Folch method. By minimizing the complex, laborious and time-consuming lipid extraction process, our method demonstrated distinct advantages for use with small scale-lipidomics in which highspeed and efficient sample preparation are required.
EXPERIMENTAL SECTION Fabrication of microchips. A silicon (Si) master mold for the poly-dimethylsiloxane (PDMS) replica was fabricated using a standard soft lithography process.24 First, SU-8 photoresist (Microchem Corp., USA) was spin-coated on a Si wafer with a thickness of 100 µm. The 45 circular-shaped, micropillar features surrounding a central hole were arrayed with three different gaps (100, 200, and 300 µm) between pillars up to three layers. The diameter of the cross section of the micropillar varied from between 200 and 560 µm, depending on the gap between the micropillars. Sylgard 184 elastomer (PDMS, Dow Corning, USA) was poured on the Si master mold with a thickness of about 4-6 mm and peeled off from the Si master after polymerization. An inner well was created using two different punches (6 and 10 mm in diameter). This donut-shaped inner well was placed into the lager holes (15 mm in diameter) made of PDMS and bonded onto another PDMS slab as a bottom layer. This assembled microchip consisted of double-layered wells. The overall dimensions of single device were about 25 × 25 mm. For a high-throughput device, we used a multi-well plate-based platform. Twenty four donut-shaped PDMS inner wells were bonded onto the glass-bottom of the well plate (well diameter = 13 mm) (MatTak Corporation, USA). Lipid extraction using SAPs. The SAPs were purchased from LG Chem, Ltd., South Korea. The powder was analyzed to measure the size of the individual SAP particles using a scanning electron microscope (JSM-6610LV, JEOL, Japan). Premixed standards, SPLASHTM, and total lipid extracts, such as porcine brain, bovine liver, and E. coli, were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA), and they were dissolved in organic solvents, such as methanol and chloroform,
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according to the provider’s instruction. The standard mixture includes all of the major lipid classes of human plasma such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), phophatidic acid (PA), lyso-phosphatidylcholine (LPC), lyso-phosphatidylethanolamine (LPE), cholesterol ester (Chol Ester), monoacylglycerol (MAG), diacylglycerol (DAG), triacylglycerol (TAG), sphingomyelin (SM), and cholesterol. The standard mixture dissolved in 100% methanol was dried to evaporate the organic solvent. Then, dried lipids were re-dissolved in a one-fourth volume of the mixture (2:1) of methyl-tert-butyl ether (MTBE) and methanol (MeOH). This solution was then re-diluted with 4 volumes of MS-grade water. The final concentration of the standard mixture was equal to the original concentration. The components and the stock concentrations of the standard lipid mixture are listed in Table S1. For the preparation of the biological samples, the concentration of brain, liver and E. coli extracts were adjusted to 5 mg mL-1 by dissolving the extracts in a 1:4 (v/v) mixture of methyl-tert-butyl ether (MTBE) and methanol (MeOH) and re-diluted with MS-grade water to a final concentration of 1 mg mL-1. Plasma (10-100 μL) was used without further dilution. For lipid extraction, 2-15 mg of SAP powder were placed into a microcentrifuge tube or the inner well of a microchip in proportion to the volume of the sample (10-100 μL). Then, the samples were dropped into SAP powder and left for 30 s to complete the gelation. Four different organic solvents, such as 2:1 (v/v) MTBE:MeOH, 2:1 (v/v) chloroform (CHCl3):MeOH, aceonitrile (ACN), and MeOH, were loaded to the gel and the samples were incubated for 2-3 min. The volume of solvent (40-400 μL) also was proportional to the volumes of the samples. The lipids were solubilized into the organic phase and extracted from the SAP gel. The solvent phase that contained the solubilized lipids was extracted using a pipette. In the case of tests in a microfluidic device, the lipids solubilized in the solvent released out from the inner well to the outer well through the gaps between the pillars that surrounded the inner well. The extracted lipids were collected into the outer well, taken with a pipette. Then, they were analyzed using direct infusion electrospray ionization quadrupole time-of-flight mass spectrometry (ESI/QTOF MS) and ultra-performance lipid chromatography-mass spectrometry (UPLC/MS) techniques. Modified Folch method. For comparison with our method, we extracted lipids from the standard mixture using the modified Folch method as described in the previous literature.13, 14 When we extracted 100-μL of sample, we added 300 μL of CH3OH and 600 μL of MTBE in that order. After mixing for 1 h, 180 μL of MS-grade water were added for phase separation followed by centrifugation at 13,000 rpm for 10 min. The upper organic phase was collected and dried with nitrogen gas. The extracted lipids were dissolved in 100 µL of CHCl3:MeOH (1:1, v/v) for storage at 4 ºC before MS analysis. Profiling and quantification of lipids by UPLC/Q-TOF MSE analysis. For the evaluation of the lipid recovery before and after lipid extraction using the SAP method and the modified Folch method, UPLC-MS analyses were used with the same amount of standard lipid mixture (10, 50 and 100 μL). To minimize volume variance after lipid extraction between the Folch and the SAP samples, all the samples were dried and re-diluted with the equal volume of buffer for LC-MS. The extracted lipids were analyzed using a Waters Synapt G2-HDMS mass spectrometer (Waters, Manchester, UK), which was operated on the MassLynx 4.1 software and coupled to the AQUITY UPLC™ system. The chromatographic separation was performed on a
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Analytical Chemistry
CSH C18 AQUITY UPLC™ column (1.7 µm, 2.1 × 100 mm; Waters, MA, USA) with the mobile phases A and B, which consisted of acetonitrile/water (60:40, v/v) and 2-propanol/acetonitrile (90:10, v/v) with 0.1% formic acid (in mobile phase A) and 10 mM ammonium formate (in mobile phase B). The buffer for mobile phase B was mixed for 3 hrs to fully dissolve and then filtered with a 0.22 μm membrane filter to remove precipitates. The flow rate and injection volume were 0.4 mL min-1 and 3 µL, respectively. The total chromatographic separation runtime was 20 min. The MS elevated energy (E) spectra were obtained in both positive-ion and negative-ion modes of ESI source. The MS conditions were optimized as follows, i.e., the capillary voltages were 2 kV and 1 kV, the cone voltage of 40 V, the source temperatures were 120 and 100 ºC, and acquisition mass range of m/z was 50 to 1200 for both ionization modes. The high-energy MS spectra were acquired alternatively with the low-energy MS spectra in UPLC/MS analysis. The collision energy used for acquiring the high- energy MS spectra was 35 eV. The high energy MS spectra provided the information about the structures of the lipids. The quantitative values of each lipid standard extracted by SAP method was compared with that by Folch method, based on the ratio of their extracted ion chromatogram (XIC) peak area in MS analysis. For the qualitative lipid profiling of the biological samples, the samples were infused directly into the electrospray source of the mass spectrometer in high resolution mode (R = 35,000) using a syringe pump, and a 0.25 mL syringe at a flow rate of 2.0 μL min-1, ESI voltage of 2.5 kV, sampling cone of 40 V, a source temperature of 120 °C, cone gas at 50 L h-1, and desolvation gas at 300 L h-1. The lipid species were identified using an in–house iLipid searching software (Ver. 1.2), which was developed at the Korea Basic Science Institute (KBSI). The lipid molecular species were observed in high-resolution (R > 30,000) mass profiling data of extracted lipids with the accuracy of 10 ppm mass tolerance. MALDI-MS analysis. In order to investigate the effect of the organic solvent on SAPs during the extraction process, 5 mg of SAPs were incubated in 200 μL of four different solvents (MTBE, chloroform, ACN, and MeOH) for 10 min. As an internal control, 30 fmoles of 1,2-diheptadecanoyl-sn-glycero-3phosphocholine (PC 17:0/17:0) (Avanti Polar Lipids, AL, USA) were spiked into the samples. One microliter of the supernatant was dropped on the target plate and mixed with α-cyano-4-hydroxycinnamic acid (CHCA) as a matrix. CHCA was dissolved in 50:50 (v/v) acetonitrile/water containing 0.5% TFA at a concentration of 10 mg mL-1. The MS spectra were obtained in a reflectron mode (R = 15,000) using a Bruker UltrafleXtremeTM matrix-assisted laser desorption/ionization (MALDI) MS instrument (Bremen, Germany) equipped with a SmartBeamTM II laser. Monitoring of the extraction of lipids using a fluorescence microscope. In order to monitor the overall process of lipid extraction in the microfluidic system, two different fluorescent lipid analogs were used, i.e., PC tagged with boron-dipyrromethene (BODIPY) FL (D3792, Thermo Fisher Scientific, Ex/Em: 503/512) and ceramide tagged with BODIPY TR (D7540, Thermo Fisher Scientific, Ex/Em: 589/617), which were purchased from Thermo Fisher Scientific (MA, USA). The fluorescent lipid analogs were dissolved in dimethyl sulfoxide (DMSO) as a stock solution with a concentration of 1 mg mL-1, and the solutions were diluted with MS-grade water to make their concentrations 0.05 mg mL-1. Five miligrams of SAP powder were used for 20-μL of sample, and then, 80 μL of solvent (2:1 (v/v) MTBE:MeOH) were added in sequence. The incubation time
with the solvent varied from 30 s to 5 min at room temperature. The overall process for extracting the fluorescent lipid analogs was monitored in real time using a fluorescence microscope (IX81, Olympus, Japan), and time-series images were acquired. To calculate the extraction efficiency depending on the incubation time and loading cycle number of solvent under SAP method, the fluorescent lipid analogs were taken before and after extraction by a pipette and analyzed with a fluorescence reader (DTX880, Beckman Coulter, USA). Up to three loading cycles of solvent were used. Monitoring of liquid flow in a microchip. In order to monitor how fast the solvent flows in a microchip during the extraction of the lipids and to determine the flow rate of solvent required for a sufficient level of lipid extraction, we measured the time until the solvent was drained completely from the inner well to the outer well. Fifty microliter of solvent (2:1 (v/v) MTBE: MeOH) were added in the inner well and the time that was spent was recorded. The flow was monitored depending on the sizes of the gaps between the pillars, i.e., 100, 200, and 300 μm, and the numbers of layers of pillars (1-3 layers). The change of the flow rate based on the different distance (1 mm and 2.5 mm) between the walls of the inner well and the outer well also was monitored. Statistical analysis. For quantitative analysis to calculate the recovery of lipids, all experiments were performed in three-replicate SAP microchips and SAP tubes at each condition and the Folch method was also performed with three-replicate samples at each condition. MS analysis was conducted once for each sample. For MALDI-MS analysis and lipid profiling, multiple experiments were performed and a representative chromatogram was obtained. A Student’s t-test was performed and asterisks indicate statistically significant differences (*p
