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Multiplexed Detection of Epigenetic Markers Using Quantum Dot (QD)-Encoded Hydrogel Microparticles Sang Yun Yeom, Choong Hyun Son, Byung Sun Kim, Sung Hyun Tag, Eunjoo Nam, Hyogeun Shin, So Hyun Kim, Haemin Gang, Hyunjoo Jenny Lee, Jungkyu Choi, Heh-In Im, Il-Joo Cho, and Nakwon Choi Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04190 • Publication Date (Web): 14 Mar 2016 Downloaded from http://pubs.acs.org on March 15, 2016

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Figure 1. Overview of multiplexed detection of modified histones using QD-encoded hydrogel microparticles. a. Schematic illustration showing fabrication of QD-encoded hydrogel microparticles via Stop Flow Lithography (SFL). Water-soluble methacrylated QDs (red, green, blue, or no color) were immobilized photochemically in PEGDA microparticles upon UV exposure. 5 reservoirs of PEGDA pre-polymer solutions were connect-ed to a PDMS microfluidic chip with 7 inlets (inlet 1: a no colored PEGDA solution; inlets 2-6: methacrylated QD-dispersed PEGDA solutions bridged with a custom 4-by-1 fluidic switch; inlet 7: a PEGDA solution dispersed with an acrylated capture antibody specific to a modified histone. Both UV LED and 2 pressure regulators were controlled by a custom-LabVIEW code. b. Schematic protocol of multiplexed epigenetic assay. 1) incu-bation with a brain lysate per mouse extracted from 3 different regions: NAc, DSt, and Cbl, 2) incubation with a biotinylated report antibody and a reporter fluorophore of SA-PE. 177x124mm (300 x 300 DPI)

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Figure 2. Characterization of QD-encoded hydrogel microparticles. a. Graph showing width of QD stripes in the code region of hydrogel microparti-cles, controlled by varying ratio of pressure to probe and code regions. For a given Pprobe/Pcode, a black circle is mean width of 25 QD stripes from 5 microparticles. Error bars represent standard deviation. b. Fluorescence micrographs of representative QD-encoded hydrogel microparticles as demonstration of 15 color-codes. c. Scatter plot indicating distributions of width of QD stripes in the 15 color-coded microparticles. Pprobe / Pcode was 3.3. Black circles and red lines are width values of 25 QD stripes (5 microparticles) and mean of the width, respectively. d. Fluorescence micrographs of representative hydrogel microparticles encoded with identical QD stripes (green and blue), consecutively located in the code region: BBBBB, GBBBB, GGBBB, GGGBB, GGGGB, and GGGGG. e. Plots showing width of both green and blue QD stripes as number of each stripe varies. Green and blue circles are mean width decoded from 5 microparticles, and error bars represent standard deviation. Gray dotted lines are linear regression curves (R2 = 0.99, slope = 14.4 for green and 14.2 for blue). 188x68mm (300 x 300 DPI)



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Figure 3. Characterization of microfluidic capture chip. a. Schematic view of microfluidic capture chip composed of a serial array of chambers and bypass channels. Individual QD-encoded hydrogel microparticles are captured in capture chambers. b. Bright field images representing sequential capture of QD-encoded hydrogel microparticles in chambers at (i) t = 0, (ii) 3.6, (iii) 4.8, and (iv) 14.8 s. c. Merged image of a fluorescence micro-graph and a bright field image showing 2 of the QD-encoded hydrogel microparticles captured in chambers. 184x66mm (300 x 300 DPI)


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Figure 4. Multiplexed relative quantification of histone H3K9 modifications directly from brain lysates of cocaine-exposed mice. a. Color-mapped fluorescence micrographs showing probe regions after multiplexed detection of Ac-H3K9, 2Me-H3K9, and 3Me-H3K9 from NAc. Left and right columns represent saline and cocaine cases respectively. b. Bar graphs presenting mean ratiometric fluorescence intensity (backgroundsubtracted) of Ac-H3K9, 2Me-H3K9, and 3Me-H3K9 simultaneously detected from (i) NAc, (ii) DSt, or (iii) Cbl per mouse. White and gray bars are ratiometric fluorescence intensity from saline- and cocaine-exposed mice for 7 days. (i) Ac-H3K9 is significantly elevated while 2Me-H3K9 is reduced in NAc, respectively indicating increase in active transcription and decrease in transcriptional repression. (ii and iii) H3K9 modification is unchanged in DSt and Cbl, which are the regions previously identified as lacking cocaineinduced H3K9 alterations and relationship with cocaine addiction, respective-ly. **** and ** denote significant differences as p values described. Error bars represent standard error of mean. Numbers of mouse used (n) were 11 (NAc), 10 (DSt-saline), 13 (DSt-cocaine), 6 (Cbl-saline), and 11 (Cbl-cocaine). Note that at least 5 QD-encoded hydrogel microparticles were analyzed for each case (i.e., histone modification-condition-brain region). c. Bar graph showing ratios of acetylation to sum of di- and trimethylation (i.e., methylation) in NAc, DSt, and Cbl. White and gray bars represent saline and cocaine cases. H3K9 acetylation/methylation ratio is increased in NAc only, demonstrating that the balance of histone H3K9 modification shifted towards gene activation. **** denotes significant difference as p val-ues described. 170x112mm (300 x 300 DPI)



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Figure 5. Relative quantification of histone H3K9 modifications from western blot analysis. a. Images of representative western blot results. b. Bar graphs presenting mean ratiometric intensity of Ac-H3K9, 2MeH3K9, and 3Me-H3K9 from western blot analysis for (i) NAc, (ii) DSt, and (iii) Cbl. **** and ** denote significant differences as p values described. Error bars represent standard error of mean. c. Bar graph showing ratios of acetylation to sum of di- and tri-methylation (i.e., methylation) in NAc, DSt, and Cbl. White and gray bars represent saline and cocaine cases. ** denotes signifi-cant difference as p values described. n ≥ 4 mice per group. 178x121mm (300 x 300 DPI)


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Quantum dot (QD)-encoded polyethylene glycol diacrylate (PEGDA) hydrogel microparticles enable multiplexed quantification of histone modifications in a single histone residue from a very small amount of brain nuclear protein lysates extracted from a cocaine-exposed mouse. This approach would greatly increase the efficiency and data quality of measuring alterations in various epigenetic markers. 130x96mm (300 x 300 DPI)



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Multiplexed Detection of Epigenetic Markers Using Quantum Dot (QD)-Encoded Hydrogel Microparticles Sang Yun Yeom1,2#, Choong Hyun Son1#, Byung Sun Kim3,4,5, Sung Hyun Tag3,4, Eunjoo Nam4, Hyogeun Shin1,6, So Hyun Kim1, Haemin Gang1, Hyunjoo J. Lee1,6, Jungkyu Choi2,8*, Heh-In Im3,4,5*, Il-Joo Cho1,6*, and Nakwon Choi1,6* 1

Center for BioMicrosystems, Brain Science Institute, Korea Institute of Science and Technology (KIST), Seoul 02792, Korea

2

Department of Chemical and Biological Engineering, Korea University, Seoul 02841, Korea

3

Convergence Research Center for Diagnosis, Treatment and Care System of Dementia, Korea Institute of Science and Technology (KIST), Seoul 02792, Korea 4

Center for Neuroscience, Brain Science Institute, Korea Institute of Science and Technology (KIST), Seoul 02792, Korea

5

Department of Neuroscience, Korea University of Science and Technology (UST), Daejeon 34113, Korea

6

Department of Biomedical Engineering, Korea University of Science and Technology (UST), Daejeon 34113, Korea

7

School of Electrical Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Korea

8

Green School, Korea University, Seoul 02841, Korea

ABSTRACT: Epigenetic alterations in gene expression are influenced by experiences and environment, resulting in significant variation of epigenetic markers from individual to individual. Therefore, it is imperative to measure various epigenetic markers simultaneously from samples of individual subjects to accurately analyze the epigenetic markers in biological samples. Moreover, the individualized genome-wide analysis has become a critical technology for recent trends in clinical applications such as early diagnosis and personalized medicine screening of numerous diseases. The array-based detection of modified histones, conventionally used for multiplexed analysis of epigenetic changes, requires pooling of samples from many subjects to analyze population-wise differences in the expression of histone markers and does not permit individualized analysis. Here, we report multiplexed detection of genome-wide changes in various histone modifications at a single-residue resolution using Quantum Dot (QD)-encoded polyethylene glycol diacrylate (PEGDA) hydrogel microparticles. To demonstrate the potential of our methodology, we present the simultaneous detection of 1) acetylation of lysine 9 of histone 3 (Ac-H3K9), 2) di-methylation of H3K9 (2Me-H3K9), and 3) tri-methylation of H3K9 (3Me-H3K9) from three distinct regions in the brain (nucleus accumbens (NAc), dorsal striatum (DSt), and cerebellum (Cbl)) of cocaine-exposed mice. Our hydrogel-based epigenetic assay enabled relative quantification of the three histone variants from only 10 μL of each brain lysate (protein content = ~1 μg/μL) per mouse. We verified that the exposure to cocaine induced a significant increase of acetylation while a notable decrease in methylation in NAc.

Epigenetic markers have the ability to generate a diverse range of phenotypes from the same genotype1 by enabling prolonged changes in postnatal traits without altering genome sequences themselves. Among the epigenetic markers, post-translational modifications of histones are fundamental to the regulation of numerous cellular processes including gene expression and DNA-protein interactions. Because aberrations in histone modifications often appear in numerous chronic diseases such as neuropsychiatric disorders,2,3 characteristic profiling of multiple modified histones facil-

itates identification of hidden genetic variations arising from chronic disorders. Drug addiction is a disorder of neuroplasticity with stable and longterm changes in brain functions.4 Drug-induced epigenetic alterations are major causes by which repeated perturbations of various signaling pathways can ultimately lead to a wide variety of stable, long-lasting changes in neuronal nuclear functions and interlinked endophenotypes. Recent epigenetic studies on the drug addiction have revealed that significant changes occur specifically in histone modifications within the brain, and such changes have been cou1


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pled with the behavioral sensitivity to addictive drugs.5 Particularly, epigenetic effects of cocaine addiction have been extensively studied.5,6 Accumulative evidence has suggested that cocaine induced increases in gene expression within nucleus accumbens (NAc), which is a reward-associated region in the brain and critically implicated in drug craving and seeking.7,8 These increases were largely attributed to overall increases in acetylation and decreases in methylation of histones.9 Therefore, identifying various changes in modified histones is imperative to gain further insight into the regulation of gene expression by the drug addiction.

modified substrate residues, MS inherently does not allow for direct detection of targets in complex samples such as cell and tissue lysates since it analyzes ionized analytes.11,12 In addition, one sample can be analyzed at a time with a complicated instrument.13 Therefore, recent interests in epigenetic modifications of histones have increasingly demanded a customizable, high-resolution assay technology capable of multiplexed epigenetic assays which can reflect the diversity of histone modifications (i.e., modification types and positions).14 For multiplexed detection of various types of biological entities such as DNA fragments15, miRNAs16, mRNAs17, and proteins18,19 in a both quantitative and robust manner, the utilization of photopatterned PEGDA hydrogel microparticles have shown great promises. More specifically, this technology allows for the fabrication of a large quantity of hydrogel microparticles lithographically defined in a polydimethylsiloxane (PDMS) microfluidic channel upon rapid cycles of ultraviolet (UV) exposures and for the application of the photocrosslinked hydrogel matrix to a variety of bioassays. This hydrogel microparticle-based approach presents four major advantages: 1) photochemical immobilization of probes such as DNA oligomers15-17, aptamers20, and antibodies18,19, 2) capability of various encoding with bar-15-19,21 or color-codes22,23 for multiplexed high-throughput bioassays, 3) high detection capacity due to the three-dimensional (3D) hydrogel matrix17, and 4) versatile combinations of the multifunctionality22-24 on demand achieved through precise projection lithography as well as microfluidic laminar coflows.

To explore the epigenetic alterations in drug addiction, conventional techniques for histone modification profiling are available such as western blot and chromatin immunoprecipitation (ChIP). Yet, these existing methods do not permit simultaneous detection of multiple modified histones directly from small amounts (< 15 μg) of brain lysates. In addition, the most vital challenges in currently available epigenetic assays are limited multiplexing capability and insufficient reliability. For instance, an amount of proteins in lysate from a region in the brain of a single mouse is too small to perform the western blot, which is inherently limited to observe ensemble effects from multiple animals. Moreover, the western blot allows only for qualitative assessment based on immunotransferred bands. The ChIP is known to produce less accurate and less reliable information due to random cuts of DNA during sonication, and to possibly lead to skewed amplification via polymerase chain reaction (PCR) with the randomly cut DNA fragments.10 Although the mass spectroscopy (MS) can serve as a powerful strategy for analyzing

Figure 1. Overview of multiplexed detection of modified histones using QD-encoded hydrogel microparticles. a. Schematic illustration showing fabrication of QD-encoded hydrogel microparticles via Stop Flow Lithography (SFL). Water-soluble methacrylated QDs (red, green, blue, or no color) were immobilized photochemically in PEGDA microparticles upon UV exposure. 5 reservoirs of PEGDA pre-polymer solutions were connected to a PDMS microfluidic chip with 7 inlets (inlet 1: a no colored PEGDA solution; inlets 2-6: methacrylated QD-dispersed PEGDA solutions bridged with a custom 4-by-1 fluidic switch; inlet 7: a PEGDA solution dispersed with an acrylated capture antibody specific to a modified histone. Both UV LED and 2 pressure regulators were controlled by a custom-LabVIEW code. b. Schematic protocol of multiplexed epigenetic assay. 1) incubation with a brain lysate per mouse extracted from 3 different regions: NAc, DSt, and Cbl, 2) incubation with a biotinylated report antibody and a reporter fluorophore of SA-PE.

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dispersed PEGDA pre-polymer solutions 1-4 were applied into 4 reservoirs. The acrylated antibody-dispersed PEGDA pre-polymer solution was applied into a 0.5 mL microtube with a cap cut off. Then, the microtube was placed on top of a bottleneck of a glass reservoir. The PEGDA pre-polymer solutions were loaded into the PDMS microchannel via tubing (OD: 0.06 in, ID: 0.02 in) and photocrosslinked upon periodic exposure of UV for 200 ms through a film photomask with a transparent opening of 1.2 mm in diameter and a filter cube set (XF02-2, Omega Optical). For the fabrication of QD-encoded microparticles used in our epigenetic assays, pressure applied to probe and code reservoirs (i.e., Pprobe and Pcode) were 100 and 30 kPa respectively. For the characterization of width of QD stripes, Pprobe : Pcode were 30:30 (1:1), 45:15 (3:1), 50:10 (5:1), 52.5:7.5 (7:1), and 54:6 (9:1) kPa. Fabricated hydrogel microparticles were rinsed and stored in 1× Phosphate-Buffered Saline (PBS; Corning) with 0.05% [v/v] Tween-20 (PBST) at 4 °C. HEK293TN Cell Culture. The human cell line HEK293TN (SBILV900A-1, Systems Biosciences) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; 11995-073, Gibco) supplemented with 10% [v/v] fetal calf serum (16000-044, Gibco) and penicillin streptomycin (15140-122, Gibco) at 37°C and 5% CO2. For the inhibition of the DNA methyltransferases (DNMT), 8×105 cells were plated per well of 6-well plates. After 48 hr, the cells were treated with 2.5 or 5.0 µM 5-aza-2'deoxycytidine (AZA; A3656, Sigma-Aldrich). For the inhibition of histone deacetylase (HDAC), 2×105 cells were plated per well. After 24 hr, the cells were treated with 50 or 100 ng/mL trichostatin A (TSA; T8552, Sigma-Aldrich). Total Protein Extraction from Cultured Cells. Media was removed and cells were washed with ice-cold PBS. Cell pellets were lysed with a lysis buffer (9803S, Cell Signaling) supplemented with Halt™ protease and phosphatase inhibitor cocktail, EDTA-free (78441, Thermo Fisher), and incubated on ice for 10 min. Then, suspensions were centrifuged at 14,000 rpm for 30 min and supernatants were collected. Protein concentration was measured with the Bio-Rad protein assay (500-0006, Bio-Rad) according to the manufacturer’s protocol. Animals. Male C57BL/6 mice weighing 21–24 g (Daehan Bio Link) were group-housed (3–4 per cage) in clear plastic cages with wire grid lids. All mice were kept on a 12 hr light/dark cycle (lights off at 7:00 AM) with access to food and water ad libitum. All procedures regarding the handling and use of animals were conducted as approved by the Institutional Animal Care and Use Committee serving the Korea Institute of Science and Technology (KIST). Cocaine Administration. Cocaine exposure procedure was based on a previous study.27 Briefly, cocaine hydrochloride (Macfarlan Smith Ltd.) was dissolved in a sterile saline solution (0.9% [w/v]). The mice received daily intraperitoneal administration of cocaine (20 mg/kg) for 7 days. A control group received intraperitoneal administration of saline daily for 7 days. Preparation of Brain Tissues. Mice were sacrificed by decapitation at 24 hr after the last cocaine administration. Fresh whole brains were isolated and stored at -70 °C. Cerebellum was isolated from the frozen whole brain with a surgical knife and briefly strored in ice for nuclear extraction. The remaining frozen brain was then microdissected to thickness of 100 μm in the coronal plane with cryostat (CM3050S, Leica Microsystems) at -20 °C. Bilaterally microdissected DSt and NAc sections were collected and further used for nuclear extraction. Nuclear Protein Extraction from Brain Tissues. Separated brain tissues were subjected to nuclear protein extraction. Briefly, cytoplasmic extraction buffer (CEB; 25 mM HEPES, 5 mM KCl, 0.5 mM MgCl2, 1× protease inhibitor mix, 1 mM PMSF, 0.5% NP-40; Sigma-Aldrich) was added to the brain tissues to final concentration of 300 mg brain per 1.0 mL CEB. The brain was homogenized, incubated in ice for 10 min, then centrifuged at 4 °C at 2,500 rpm for 5 min. After supernatant was gently discarded, 200 μL of CEB was added and this mixture was centrifuged at 4 °C at 2,500 rpm for 5 min, and the supernatant was discarded again. Subsequently, nuclear extraction buffer (25 mM HEPES, 10% sucrose, 350 mM NaCl, 1× protease inhibitor mix, 1 mM PMSF, 0.01% NP-40; Sigma-Aldrich) was added to the remaining pellet and mixed by vortexing. The mixture was

Here, we report, for the first time to our knowledge, multiplexed detection of genome-wide changes in various modified histones from small amount of nuclear protein lysates, with each lysate extracted from a single mouse, using Quantum Dot (QD)-encoded polyethylene glycol diacrylate (PEGDA) hydrogel microparticles. Specifically, we present characterizations of 1) QDs photochemically immobilized within PEGDA microparticles as an encoding platform and 2) a microfluidic chamber array to capture individual hydrogel microparticles as a platform for both decoding and signal detection. Then, we demonstrate the simultaneous detection of 1) acetylation of lysine 9 of histone 3 (Ac-H3K9), 2) di-methylation of H3K9 (2Me-H3K9), and 3) tri-methylation of H3K9 (3MeH3K9) from three distinct regions in the brain (NAc, dorsal striatum (DSt), and cerebellum (Cbl)) of cocaine-exposed mice to identify epigenetic effects of chronic exposure to cocaine in the context of drug addiction.

EXPERIMENTAL SECTION Fabrication of QD-Encoded Hydrogel Microparticles Using Computer-synchronized Pressure-UV. The fabrication of PEGDA hydrogel microparticles was performed via SFL similarly as described in previous work16,17 (Fig. 1a). Briefly, 4 major differences were applied for the fabrication in this study. First, water-soluble methacrylated QDs (red: MagQuTM-91002 Qdot-615 nm-Methacrylate; green: MagVigenTM Customize-61002 GA-Qdot-535 nm-Methacrylate; blue: MagQuTM91002 Qdot-460nm-Methacrylate, NVIGEN) were immobilized photochemically in PEG700DA matrices upon UV exposure. Second, a light source was collimated UV (365 nm) LED for Zeiss Axioskop (M265L2-C4, Thorlabs). Third, 5 reservoirs of PEGDA pre-polymer solutions were connected to a 60 μm-high PDMS (Sylgard 184, Dowhitech Silicone Co.) microchannel with 7 inlets (inlet 1: a no colored PEG700DA pre-polymer solution; inlets 2-6: methacrylated QD-dispersed PEGDA pre-polymer solutions bridged with a custom 4-by-1 fluidic switch; inlet 7: a PEGDA pre-polymer solution dispersed with an acrylated capture antibody specific to a modified histone. Fourth, both a UV LED driver (LEDD1B T-Cube LED driver, Thorlabs) and 2 pressure regulators operated by electrical voltage (one for the code region and the other for the probe region; ITV0031, SMC Korea) were controlled by a custom-LabVIEW (National Instruments) code via a data acquisition (DAQ) board (CDAQ-9171, National Instruments) and an analog voltage output module (NI 9264, National Instruments). 5 PEGDA pre-polymer solutions were prepared for the code and probe regions. PEGDA pre-polymer mixture 1-4 consisted of 40% [v/v] PEG700DA (Sigma-Aldrich), 20% [v/v] PEG600 (Sigma-Aldrich) porogen, 5% [v/v] Darocur 1173 photoinitiator (Sigma-Aldrich), 7.5% [v/v] deionized (DI) water, and 27.5% [v/v] QD solution (red, green, blue, and DI water for no color). PEGDA pre-polymer mixture 5 consisted of 20% [v/v] PEG700DA, 40% [v/v] PEG600, 5% [v/v] photoinitiator, and 35% [v/v] 3× Tris-EDTA (TE) buffer (Sigma-Aldrich). Acrylation of anti-acetyl histone H3K9 (ab12179, Abcam), di-methyl histone H3K9 (ab1220, Abcam), and tri-methyl histone H3K9 (ab8898, Abcam) was performed by mixing each antibody stock solution with an acrylation linker (Acryl-PEG-SVA; MW 2,000; 50 μg/μL; Laysan Bio) at a volume ratio of 4:1 under 3 hr with agitation of 1000 rpm at 25 °C. We note that we chose these antibodies based on an online database25 and a report that assessed the quality of histone-modification antibodies.26 After the acrylation of the antibodies, dialysis was conducted with mini dialysis devices (Slide-A-LyzerTM MINI Dialysis Device; cat. # 69570; MW cut off 10,000; Thermo Fisher Scientific) for 10 min. Then, a 1:9 [v/v] ratio of each dialyzed acrylate-antibody solution was mixed with the PEGDA prepolymer mixture 5 immediately before the fabrication of microparticles. Containers of 5 reservoirs shown in Fig. 1a were 4 mL glass vials. On each plastic cap of the vial, 2 holes were created to insert Tygon tubing (OD: 0.06 in, ID: 0.02 in). Then, an epoxy glue (ITW consumer) was applied around the tubing on the cap for air-tight sealing. The QD-

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incubated in ice for 30 min, with gentle vortexing at an interval of 10 min. Then the mixture was centrifuged at 4 °C at 14,000 rpm for 10 min and the supernatant containing nuclear protein was immediately used for Bradford assay using Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad) to quantify the amount of proteins. Then the nuclear protein was stored in 80 °C until the subsequent analyses. Total Protein Extraction from Brain Tissues. Separated brain tissues were subjected to total protein extraction. Briefly, brain tissues were homogenized in N-PER™ neuronal protein extraction reagent (87792, Thermo Fisher) supplemented with Halt™ protease and phosphatase pnhibitor cocktail, EDTA-free (78441, Thermo Fisher), and incubated on ice for 10 min. Then, suspensions were centrifuged at 10,000 rpm for 10 min and supernatants were collected. Protein concentration was measured with the Bio-Rad protein assay (500-0006, Bio-Rad) according to the manufacturer’s protocol. Western Blot. 25 µg (HEK293TN cell lysate) or 50 µg (mouse brain lysate) protein was loaded on a 12% [w/v] polyacrylamide gel together with Xpert 2 prestained protein marker (p8502, GenDEPOT) and run at 110 V for 2 hr in a running buffer (25 mM Tris base, 190 mM glycine, 0.1% [w/v] sodium dodecyl sulfate (SDS), pH ~8.3). Protein was then transferred onto Millex-HV filter, pore size of 0.45 μm, polyvinylidene fluoride (PVDF; SLHV033RS, Millipore) in a transfer buffer (25 mM Tris, 192 mM glycine, 10% [v/v] methanol) for 1 hr at 100~110 V. Then the membrane was blocked for 30~60 min in 3% [w/v] bovine serum albumin (BSA) in Tris-buffered saline (25 mM Tris, 150 mM NaCl, 2 mM KCl, pH 7.4, 15 mM NaCl, 10 mM Tris, pH 8.0) containing 0.1% [w/v] Tween-20. The membrane was incubated with primary antibodies rabbit polyclonal anti-histone H3 (tri methyl K9) (ab8898, Abcam; diluted to 1:1000), rabbit monoclonal anti-histone H3 (di methyl K9) (ab1220, Abcam; diluted to 1:1000) , mouse polyclonal anti-histone H3 (acetyl K9) (ab4441, Abcam; diluted to 1:1000), and mouse polyclonal anti-β-actin (sc-47778, Santa Cruz; diluted to 1:500) mixed in 3% [w/v] BSA in Trisbuffered saline. After incubation on a shaker at 4°C for 16 hr, the membrane was washed 3 times for 30 min in tris-buffered saline containing Tween-20 and further incubated with a secondary antibody: donkey antimouse IgG-HRP (sc-2318, Santa Cruz; diluted to 1:5000) or donkey antirabbit IgG-HRP (sc-2317, Santa Cruz; 1:5000). The membrane was finally washed 3 times for 30 min in tris-buffered saline containing Tween-20. Blots were developed by SuperSignal West Pico Chemiluminescent Substrate (34080, Thermo Fisher). Blots were detected by Bio-Image Analysis System (LAS4000, GE Healthcare Bio-Science). ImageJ and Scion Image software were used for image analysis and densitometry. Readings were corrected by background intensity and normalized to an internal control, β-actin. Multiplexed Detection of Modified Histones with QD-Encoded Hydrogel Microparticles. 3-plex detection of the modified histones was performed similarly as described in previous work18,19 (Fig. S1). 1) 10 μL of 1 μg/μL brain lysate from the nuclear protein extraction was mixed with 10 μL of 1× PBST in 500 µL microtube and vortexed for 40 s to enhance dispersion of viscous lysates. 2) After spinning down the microtube for 1-2 s, 10 μL of each microparticle suspension (~2 particle/μL in PBST) was applied to the microtube such that a total of ~60 particles (~20 particles per target modified histone) were added in a total assay volume of 50 µL. 3) Lysates spiked in assay tubes were incubated at 25 °C for 18 hr with agitation at 700 rpm. Modified histone-bound hydrogel microparticles were rinsed with 500 µL of 1× PBST 3 times. 4) 2.5 µL of 0.1 µg/µL reporter antibody (histone H3 antibody conjugated with biotin; NB500-267B; Novusbio) in PBS was added to assay tubes and incubated at 25°C for 3 hr at 700 rpm. Reporter antibody-bound hydrogel microparticles were rinsed with 500 µL of 1× PBST 3 times. 5) 3 µL of 100 ng/µL streptavidin-phycoerythrin (SA-PE) (S866; Life technology) was spiked into assay tubes and incubated at 25 °C for 30 min at 700 rpm. 6) Finally assay tubes were rinsed 500 µL of 1× PBST 3 times and then SA-PE-bound hydrogel microparticles were re-suspended in PTET (5× TE

with 25% [v/v] PEG400 and 0.05% Tween-20) for decoding and signal detection. Note that in all rinsing steps, temperature was maintained at 4 °C to prevent undesired degradation of DNA-bound histones. Fabrication of Array of Microfluidic Chambers and Bypass Channels. The microfluidic capture chip was fabricated via typical soft lithography with PDMS and a 60 µm-thick SU-8 (SU-8 50; MicroChem Co.) master. In- and outlets through PDMS replica were created by a bluntend syringe needle (17G; ~1.5 mm in diameter). Then, clean PDMS replica was covalently bonded on a glass slide immediately after O2 plasma treatment. Width of the capture chamber was 80 µm, length of the narrow (capture) channel was 270 µm, and width and pass length of the bypass channel was 165 and 1,906 µm, respectively (Fig. 3a). Image Acquisition and Analysis. The QD-encoded hydrogel microparticles suspended in PTET were captured in the array of microfluidic chambers, on an inverted fluorescence microscope (Axio Observer.A1, Zeiss). An LED lamp (SOLA SM II, Lumencor Light Engine) was used as a white light source and filtered through a cube set (XF02-2, Omega Optical) for imaging of the code region or a cube set (89101x, 89101m, 89100bs, Chroma Technology Co.) for imaging of the probe region. 8-bit RGB and 16-bit monochrome fluorescence images were acquired using a digital single-lens reflex (DSLR) camera (EOS 6D, Cannon) with exposure time of 1-2 s and a scientific complementary metal–oxide–semiconductor (sCMOS) camera (optiMOS, QImaging) with exposure time of 100 ms, respectively. Both cameras were connected to the microscope via a dual adapter. Acquired images with a 20× objective lens were then rotated, cropped, and analyzed using a custom code (Matlab, MathWorks) in a semi-automated manner (Supporting Information). Fluorescence intensity in the cropped area (550 × 250 pixel; 170 × 77 µm) covering the probe region was averaged for each hydrogel microparticle. Background signal was defined as mean fluorescence intensity from microparticles processed through our epigenetic assay without lysates. Background-subtracted mean fluorescence intensity was then calculated from at least 5 hydrogel microparticles. Ratiometric intensities were calculated by dividing backgroundsubtracted mean fluorescence intensity of each modified histone (AcH3K9, 2Me-H3K9, or 3Me-H3K9) from sum of all the intensities (e.g., IAcH3K9/(IAc-H3K9 + I2Me-H3K9 + I3Me-H3K9)). Statistical Analysis. The ratiometric intensities and normalized fold changes were used for statistical analyses in Prism (GraphPad). Statistical significance was assessed using multiple t-tests (Figs. 4b, 4c, 5b, 5c, and S7) and 1-way ANOVA (Fig. S8). For multiple t-tests, p values are noted in the figures. For 1-way ANOVA, confidence level was 95%, and **** denotes p < 0.0001.

RESULTS AND DISCUSSION Figure 1 shows a schematic overview of our approach: 1) fabrication of QD-encoded hydrogel microparticles (Fig. 1a), and 2) multiple 3-plexed epigenetic assays to detect Ac-H3K9, 2Me-H3K9, and 3Me-H3K9 that were present in each lysate of NAc, DSt, or Cbl in the brain of a single mouse exposed to cocaine for 7 days (Fig. 1b). Our hydrogel-based epigenetic assay enabled relative quantification of the three histone variants from only 10 μL of each brain lysate (protein content = ~1 μg/μL) per mouse. Characterization of hydrogel microparticles encoded with QD stripes. As an encoding platform for multiplexing, we incorporated color-codes with red (615 nm), green (535 nm), and blue (450 nm) QDs within PEGDA microparticles. QDs are nanocrystals that emit distinct visible colors determined by the size of the crystals. As the QD size decreases, the energy difference between valence and conduction bands (i.e., band gap energy) increases in an almost discrete manner; therefore, colors of smaller QDs shift from red towards blue. A practical advantage of QDs for colorcodes is that end users need only a single excitation wavelength (typically UV; 330-360 nm) for simultaneous imaging of multiple 4



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Figure 2. Characterization of QD-encoded hydrogel microparticles. a. Graph showing width of QD stripes in the code region of hydrogel microparticles, controlled by varying ratio of pressure to probe and code regions. For a given Pprobe/Pcode, a black circle is mean width of 25 QD stripes from 5 microparticles. Error bars represent standard deviation. b. Fluorescence micrographs of representative QD-encoded hydrogel microparticles as demonstration of 15 color-codes. c. Scatter plot indicating distributions of width of QD stripes in the 15 color-coded microparticles. Pprobe / Pcode was 3.3. Black circles and red lines are width values of 25 QD stripes (5 microparticles) and mean of the width, respectively. d. Fluorescence micrographs of representative hydrogel microparticles encoded with identical QD stripes (green and blue), consecutively located in the code region: BBBBB, GBBBB, GGBBB, GGGBB, GGGGB, and GGGGG. e. Plots showing width of both green and blue QD stripes as number of each stripe varies. Green and blue circles are mean width decoded from 5 microparticles, and error bars represent standard deviation. Gray dotted lines are linear regression curves (R2 = 0.99, slope = 14.4 for green and 14.2 for blue).

colors using a long-pass emission filter. Compared to the earlier work by Han, et al.28, where they incorporated mixtures of hydrophobic QDs into swollen polystyrene microbeads, we employed QDs functionalized with both amine and methacrylate groups. These functional groups not only enhance dispersion of QDs in water as well as PEGDA pre-polymer solutions due to high affinity of the amine group to water but also enable photochemical immobilization of QDs on PEGDA crosslinks upon UV exposure via covalent conjugation between the methacrylate and acrylate groups. Furthermore, we incorporated 5 QD stripes in each hydrogel microparticle (150 μm in diameter, 55~56 μm in thickness) for simpler decoding to identify color-codes without additional analysis of individual intensities of red, green, and blue channels.

to aspect ratios of 1.6, 2.7, and 10 respectively. All of these microparticles maintained the lying orientation. We chose 10 pg/mL as the lowest yet detectable concentration of IL-2 for the microparticles with various thickness. Our data suggest that 55 μm was optimal thickness (aspect ratio = ~2.7) (Fig. S2). To generate the QD stripes in a code region of the hydrogel microparticles, we set up 4 universal reservoirs of PEG700DA prepolymer solutions (i.e., no colored (N), red (R), green (G), and blue (B) QD-PEGDA solutions) and applied the same pressure (Pcode) to the 4 reservoirs in order to achieve identical width of the QD stripes. We placed 5 independent fluidic switches to connect each reservoir with one of inlets 2 to 6 of a 7-inlet PDMS microfluidic chip. This independent switching allowed for selective control over the color for each QD stripe. With this approach, a maximum number of combinations of color-codes is equal to 1,024; (number of possible colors for each QD stripe)(number of stripes) = 45. Figure 1a illustrates an example of the proposed operation for a color-code, GBRGN, reading from the inlet 2 through 6. We used inlet 1 for an inert margin stream by delivering a no-colored PEGDA solution on purpose. In addition, we placed a no-colored stripe, which served as an inert spacer as well, next to the probe region in order to prevent potential interference between the code and probe regions. For the probe region, we applied Pprobe to a separate reservoir of a PEGDA pre-polymer solution containing an acrylate-conjugated antibody that was supposed to capture a modified histone. Similar to the QDs, the capture antibody was photochemically immobilized on PEGDA crosslinks upon UV exposure. We created a home-made circuit board to control both Pcode and Pprobe independently and to control a UV light emitting diode (LED) (365 nm) (see Fig. S3 for more detailed information about our design of the circuit board). We adopted typical sequences used in Stop Flow Lithography (SFL)16,17 (i.e., stop flow - expose UV - resume flow) and repeated the sequences to fabricate QD-encoded hydrogel microparticles.

We would like to emphasize that we optimized the thickness of hydrogel microparticles for reliable signal detection with the given diameter. Because depth-integrated fluorescence intensity acquired from hydrogel microparticles is typically used as detected signal, the thickness of microparicles is essentially associated with sensitivity. Since the number of probes photochemically incorporated within hydrogel is proportional to its volume, thicker microparticles are desirable to acquire higher fluorescence intensity from given amount or concentration of targets. However, the aspect ratio of microparticles (i.e., diameter/thickness) also serves as a practically important parameter because this aspect ratio is directly related to the orientation of hydrogel microparticles during both assays and signal detection. For example, when aspect ratios are smaller than 2 (i.e., higher thickness; analogous to rods), the sidewall of microparticles increasingly faces up (‘standing’ orientation). When, aspect ratios are larger than 10 (i.e., smaller thickness; analogous to disks), these thin microparticles are highly likely to lie (‘lying’ orientation) or to be flipped due to their flexibility. In order to determine optimal thickness of hydrogel microparticles for this study, we measured fluorescence signal from 10 pg/mL of interleukin-2 (IL2) by varying thickness (15, 55, and 95 μm), which corresponded 5


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Figure 3. Characterization of microfluidic capture chip. a. Schematic view of microfluidic capture chip composed of a serial array of chambers and bypass channels. Individual QD-encoded hydrogel microparticles are captured in capture chambers. b. Bright field images representing sequential capture of QD-encoded hydrogel microparticles in chambers at (i) t = 0, (ii) 3.6, (iii) 4.8, and (iv) 14.8 s. c. Merged image of a fluorescence micrograph and a bright field image showing 2 of the QD-encoded hydrogel microparticles captured in chambers.

We confirmed our controllability over the width of the QD stripes by varying ratios of Pcode to Pprobe (i.e., Pcode / Pprobe). Figure2a shows that we were able to control the mean width of each QD stripe from 21 ± 1.4 (upper limit) μm to 6.3 ± 0.81 (lower limit) μm as Pcode / Pprobe increases from 1 to 9 (see detailed information in Table S1). Accordingly, the ratio of the width of the probe region to that of the code region (i.e., wprobe / wcode; schematic inset image in upper right of Fig. 2a) in changed from 0.43 to 3.8. We observed that wprobe and wcode were almost the same when Pcode / Pprobe was close to 3 (wQD stripe = 15.2 ± 1.080 and 14.5 ± 1.12 when Pcode / Pprobe = 3 and 3.3, respectively). At this point, we would like to mention that the narrower QD stripes would cause two undesired consequences toward the achievement of high-throughput decoding. First, decoding time could take longer because microparticle images should be acquired at higher magnification. Second, errors in identification of the QD stripes with their width propagate as coefficient of variation (CV) of the width increases (Table S1). However, too wide width of the code region is not desirable, either, because it inevitably results in narrowing down the probe region. Considering this trade-off between reliable characterizations of the probe and code regions, we chose Pcode / Pprobe of 3.3 to keep the size of both regions almost identical. We also confirmed that variations of wQD stripe within a microparticle as well as over multiple microparticles were < ~2 μm (Fig. S4). This result indicates that our fabrication method was reliable and reproducible, and thus a large encoding capacity is highly expected through combinations of the colors in the QD stripes (theoretically up to 1,024 in this study). Figure 2b shows a fluorescence image where we randomly chose 15 types of colorcodes: GBRBG, RGBGR, BGBRG, BRGRB, GRBGB, BGRBG, RBGRG, BGRBR, GBRGB, RBRBG, NGRBG, BNGRB, GBNBR, GBRNR, and GBGRN. As expected from Fig. 2a, Pcode / Pprobe of 3.3 led to mean wQD stripe of 15 ± 0.73 μm and variations of wQD stripe (i.e., wQD stripe,max - wQD stripe,mean and wQD stripe,mean - wQD stripe,min) were less than 1.3 μm (Fig. 2c).

(Figs. 2d and 2e). The width of both green and blue stripes changed linearly as the number of the stripes varied (R2 = 0.99), and slopes of the linear regression curves were 14.4 (green) and 14.2 (blue) μm/number of green or blue stripe, which shows an excellent agreement with Fig. 2a, 2c and Table S1. Furthermore, a qualitative assessment would also allow for distinguishing the six color-codes (Fig. S5). These data provide strong evidence to ensure the decoding accuracy. CHARACTERIZATION FOR MICROFLUIDIC CAPTURE OF HYDROGEL MICROPARTICLES. In order to realize efficient both decoding and signal detection with a large number of hydrogel microparticles (e.g., at least ~20 particles per modified histone; a total of ~60 particles out of 1 assay tube for 3 different types of QDencoded hydrogel microparticles), we fabricated an array of microfluidic chambers to capture hydrogel microparticles hydrodynamically (Fig. 3a). Although typical static imaging (i.e., imaging of hydrogel microparticles between glass slides) is effective for preliminary screening or small-scale tests, we found that time taken to find appropriate microparticle images dramatically increased with the number of subsets (e.g., typically 7 assay tubes representing each brain region with controls). This undesirable, tedious task is mainly attributed to a fact that we need to move sample specimens rather in a random manner, while excluding microparticle images that show optical artifacts due to particle-particle aggregation (or contact) and particle-air interface near edges of the glass slides. To overcome this issue, we utilized hydrodynamic resistance in microfluidic chambers and bypass channels29 to permit a spatially uniform distribution of hydrogel microparticles in an array form. We designed appropriate chambers for placing the disk-shaped QD-encoded PEGDA microparticles via the effective control over the ratio of hydrodynamic resistance and input pressure. Specifically, a total of 120 capture units were connected serially, and each unit consisted of a capture chamber, a bypass channel, and a narrower channel connected to a next unit (Fig. 3a and 3b). We considered 2 fluidic paths; 1) a narrower (capture) channel between 2 adjacent capture units and 2) a D-shaped loop (bypass) channel. Hydrodynamic resistance of the narrower channel should be smaller than that of the bypass channel so that a vacant capture chamber is always occupied by a single microparticle that travels first to a capture unit. We designed a ratio of a flow rate through the capture channel to that through the bypass channel (Qcapture / Qbypass) to be

In order to expand encoding/decoding capacity, we fabricated hydrogel microparticles encoded with identical green and blue stripes consecutively located in the code region: GGGGG, GBBBB, GGBBB, GGGBB, GGGGB, and BBBBB. Since we confirmed controllability over the width of QD stripes in a very reliable manner, we were able to decode all the 6 color-codes successfully, including potentially ambiguous color-codes of GGBBB and GGGBB 6



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Figure 4. Multiplexed relative quantification of histone H3K9 modifications directly from brain lysates of cocaine-exposed mice. a. Color-mapped fluorescence micrographs showing probe regions after multiplexed detection of Ac-H3K9, 2Me-H3K9, and 3Me-H3K9 from NAc. Left and right columns represent saline and cocaine cases respectively. b. Bar graphs presenting mean ratiometric fluorescence intensity (background-subtracted) of Ac-H3K9, 2Me-H3K9, and 3Me-H3K9 simultaneously detected from (i) NAc, (ii) DSt, or (iii) Cbl per mouse. White and gray bars are ratiometric fluorescence intensity from saline- and cocaine-exposed mice for 7 days. (i) Ac-H3K9 is significantly elevated while 2Me-H3K9 is reduced in NAc, respectively indicating increase in active transcription and decrease in transcriptional repression. (ii and iii) H3K9 modification is unchanged in DSt and Cbl, which are the regions previously identified as lacking cocaine-induced H3K9 alterations and relationship with cocaine addiction, respectively. **** and ** denote significant differences as p values described. Error bars represent standard error of mean. Numbers of mouse used (n) were 11 (NAc), 10 (DSt-saline), 13 (DSt-cocaine), 6 (Cbl-saline), and 11 (Cbl-cocaine). Note that at least 5 QD-encoded hydrogel microparticles were analyzed for each case (i.e., histone modification-condition-brain region). c. Bar graph showing ratios of acetylation to sum of di- and tri-methylation (i.e., methylation) in NAc, DSt, and Cbl. White and gray bars represent saline and cocaine cases. H3K9 acetylation/methylation ratio is increased in NAc only, demonstrating that the balance of histone H3K9 modification shifted towards gene activation. **** denotes significant difference as p values described.

2.45. Figure 3b shows temporal snapshots of 7 microparticles (~15 particle/10 μL) captured sequentially from relative time of 0 to 14.8 s. Input pressure of ~25 kPa was enough to capture hydrogel microparticles in the chambers sequentially; capture efficiency, defined as a ratio of the number of microparticles captured in chambers to that introduced to the inlet of the capture chip, was ~95%. By loading over 100 microparticles, we could confirm that the microparticles always bypassed around captured chambers, not vacant ones (Fig. 3b and Video S1). Notably, time required to occupy all the chambers strongly depended on both the number density of a microparticle suspension and the input pressure. Higher values of both factors led to reducing the time. Figure 3c shows representative QD-encoded microparticles with the same colorcode captured in chambers. This approach of imaging hydrogel microparticles uniformly distributed in our capture chip is promising for efficient decoding as well as signal detection in a more reliable way through prompt analyses of a number of microparticles at a larger scale, as compared to the typical static imaging.

tification of Ac-H3K9, 2Me-H3K9, and 3Me-H3K9, respectively, from separate brain regions (i.e., NAc, DSt, and Cbl) of cocaineexposed mice. Because concentration of modified histones in the protein lysates significantly vary with individual mice, the quantification with collective samples from several mice (order of tens to hundreds microliters of lysates) cannot distinguish relatively small changes in modified histones in a reliable way unless phenotypically identical mice were sacrificed. Indeed, we initially observed random differences (data not shown here) in both acetylation and methylation of H3K9, when we performed the epigenetic assay with collective lysates obtained from several cocaine-exposed mice (Fig. S6). Therefore, we analyzed ratiometric fluorescence intensities of the probe region per mice for normalization and fair comparisons. It has been reported that the H3K9 residue in NAc is characterized by chronic cocaine-induced increases in acetylation (AcH3K9) and decreases in di-methylation (2Me-H3K9), while trimethylation (3Me-H3K9) is either unchanged or decreased.27,30-32 Color-mapped probe regions, as graphical representatives of Ac-, 2Me-, and 3Me-H3K9 detected in NAc, support this trend (Fig. 4a). The non-uniformity of color-mapped images in Fig. 4a can be attributed to mass transfer limitation in three consecutive heterogeneous (i.e., solution to gel) biochemical reactions for detection;

MULTIPLEXED DETECTION OF MODIFIED HISTONES ASSOCIATED WITH COCAINE ADDICTION. We used 3 types of QD-encoded hydrogel microparticles (color-codes of BRGBN, GBRGN, RGBGN) to demonstrate the multiplexed relative quan7


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Figure 5. Relative quantification of histone H3K9 modifications from western blot analysis. a. Images of representative western blot results. b. Bar graphs presenting mean ratiometric intensity of Ac-H3K9, 2Me-H3K9, and 3Me-H3K9 from western blot analysis for (i) NAc, (ii) DSt, and (iii) Cbl. **** and ** denote significant differences as p values described. Error bars represent standard error of mean. c. Bar graph showing ratios of acetylation to sum of di- and tri-methylation (i.e., methylation) in NAc, DSt, and Cbl. White and gray bars represent saline and cocaine cases. ** denotes significant difference as p values described. n ≥ 4 mice per group.

1) binding of modified histones (this can be considered as proteins or antigens in the general implication) in the solution phase onto capture antibodies in the gel phase, 2) binding of biotinylated report antibody onto the modified histones, and 3) binding of SA-PE onto the report antibody. We already confirmed uniform immobilization of antibodies in PEGDA hydrogel by applying SA-PE in microparticles incorporated with the biotinylated antibody (data not shown), similarly as confirmed in our previous study.17 In accordance with previous reports,27,30-32 our analysis of mean ratiometric fluorescence intensity showed considerable elevation of Ac-H3K9 and reduction of 2Me-H3K9 in NAc after repetitive cocaine exposure for 7 days along with a slight change in 3Me-H3K9 (Fig. 4a and 4b i). In contrast, H3K9 modifications in DSt and Cbl remained unchanged (Fig. 4b ii and iii, respectively), which is also in agreement with previous studies.27,33 It is well-known that the acetylation and di-/tri-methylation of H3K9 are mutually exclusive.34,35 Therefore, we used a relative ratio of acetylation to methylation to evaluate the dynamic balance of modified histones in this residue. As shown in Fig. 4c, this ratio closely resembles the global histone modification state in NAc H3K9 (Fig. 4b i), and the balance shifts towards acetylation after the repetitive cocaine exposure. These findings suggest that the balance of acetylation and di/tri-methylation can be a distinct epigenetic determinant to the chronic gene activation or repression status of NAc.

of protein (50 µg protein per one histone modification) for each brain region, which required pooling of each brain region from at least 2 mice. We first analyzed absolute quantification of each histone modification and found significantly increased Ac-H3K9 and decreased 2Me-, 3Me-H3K9 in NAc only (Fig. 5a and Fig. S7), which are in accordance with the previous reports27,30-32. Then we analyzed ratiometric analysis for each histone modification and found large increase in Ac-H3K9 and reduction in 2Me-H3K9, but no change in 3Me-H3K9 of NAc or other brain regions (Fig. 5b). Furthermore, we found that the ratio of acetylation to methylation increased in NAc whereas remained unchanged in DSt and Cbl (Fig. 5c). These results are in line with the results from our assay (Fig. 4), suggesting that the sensitivity (true positive rate) of our assay is comparable to that of western blot. Overall, not only did we demonstrate the feasibility of our method through observation of the expected results (i.e., H3K9-containing genetic loci within NAc would result in overall activation after chronic exposure to cocaine), but also 3 modified histones were simultaneously detected in our system from small amount of the protein lysates, even with each lysate extracted from small brain regions of a single mouse. Diverse types of histone modification exist in nature,14 where many are bound by mutual relationships and have inter-/intra-individual variability.34-37 To conduct genome-wide study of histone modification, measuring the relative changes between different but related types of histone modification may be essential. Our ratiometric analysis in regards to the mutually exclusive modifications of H3K9 residue (acetylation and methylation) and the resulting interpretations are highly consistent with the previous reports.

Next, we performed quantification of histone H3K9 modifications with western blot to compare the results from our assay with a conventional method. It is important to note that our assay was multiplexed quantification from 10 µg of protein from each brain region, whereas western blot was single-plexed quantification from 150 µg 8



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MULTIPLEXED DETECTION OF MODIFIED HISTONES FROM CELL CULTURE TREATED WITH HISTONE-MODIFYING DRUGS. Finally, to confirm that our assay can accurately analyze various types of biological samples in a versatile manner, we performed quantification of histone H3K9 modifications from HEK cells treated with trichostatin A (TSA), a HDAC inhibitor, or 5Aza-2’-deoxycytidine (AZA), a DNMT inhibitor. The identical 3 types of QD-encoded hydrogel microparticles (color-codes of BRGBN, GBRGN, RGBGN) were used for detecting Ac-H3K9, 2Me-H3K9, and 3Me-H3K9, respectively. As expected, western blot demonstrated significant elevation of Ac-H3K9 in TSA-treated cells and significant reduction of 2Me- and 3Me-H3K9 in AZAtreated cells (Fig. S8a). Consistently, our assay with QD-encoded hydrogel microparticles also revealed the same significant changes (Fig. S8b). These findings additionally confirm the sensitivity of our assay to changes in histone modifications and demonstrate the versatility with various biological samples.

Detailed assay scheme for multiplexed detection of modified histones from mouse brain lysates; optimization of thickness of hydrogel microparticles; circuit design to control pressure regulators and UV LED; width of QD stripes by controlling Pprobe and Pcode; inter- and intraparticle variations of width of QD stripes; spatial intensity profiles of representative microparticles encoded with identical green and blue stripes consecutively located in the code region; representative detection of H3K9 modifications from mixture of multiple brain lysates; Western blot analysis (normalized fold change) from the brain lysates of cocaine-exposed mice; quantification of histone H3K9 modifications from lysates of HEK cells treated with HDAC inhibitor or DNMT inhibitor; MATLAB code to rotate and crop acquired images; MATLAB code to analyze fluorescence intensity from (rotated and cropped) images (pdf) Video clip showing microfluidic capture of hydrogel microparticles (mp4)

Interestingly, we observed difference in changes between western blot and our system; for instance, in in vivo samples, ~20% ratiometric intensity change in histone modifications (i.e., Ac- and 2Me-H3K9) was detected by western blot, whereas ~5% change was detected by our assay. This difference could be attributed to procedural differences such as the different amount of proteins used for each assay, or separation of protein by size in western blot. In the former case, less amount of protein used in immunoassays usually makes it difficult to detect the potential difference between samples due to limiting factors such as detection threshold, impurity of protein sample (less protein used in same assay volume), etc. In this regard, our hydrogel microparticle-based assay exhibits sensitivity that is sufficiently high enough to distinguish changes in epigenetic modifications from small protein amount. In the latter case, western blot includes many steps to enhance specificity such as electrophoresis to separate proteins by size to isolate proteins in target size, blocking to prevent non-specific binding of antibodies, and enzymatic signal amplification with the secondary antibody, IgG-HRP (note that we directly image a biotinylated secondary antibody conjugated with SA-PE without signal amplification. In this aspect, integration of these steps in our assay will further improve specificity and sensitivity.

Corresponding Authors

AUTHOR INFORMATION *E-mail: [email protected] *E-mail: [email protected] *E-mail: [email protected] *E-mail: [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. #These authors contributed equally as co-first authors. *These authors contributed equally as co-corresponding authors.

ACKNOWLEDGMENT This work was supported by the KIST Institutional Program (project no. 2E26180 and 2E26664). This research was also supported by the Brain Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2012M3C7A1055410). This work was also supported by the Human Resources Development Program (No. 20134010200600) of the Korea Institute of Energy Technology Evaluation and Planning (KETEP) grant funded by the Korea government Ministry of Trade, Industry and Energy.

REFERENCES

CONCLUSIONS

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In summary, we demonstrated that PEGDA hydrogel microparticles were successfully encoded by the precisely controllable and reliable incorporation of QD stripes. The QD-encoded PEGDA hydrogel microparticle were capable of quantifying multiple modified histones in a single residue from small amount of nuclear protein lysate, with expected reproduction of cocaine-mediated epigenetic alterations in NAc. We also showed the applicability of our microfluidic capture chip for efficient decoding and signal detection. With the potential high-throughput, high-resolution capability, and low variability in interpretation, we expect that our hydrogel microparticle-based platform would greatly increase the efficiency and data quality of measuring alterations in various epigenetic markers.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI:

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Multiplexed Detection of Epigenetic Markers Using ... - ACS Publications

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