The microchip pattern was designed with AutoCAD (Autodesk). Each chip consists of 14 identical cell-scattering and deformation zones, and each zone contains 10 arrays of constrictions. The constriction depth is 15 μm, and the width varies from 4 to 5 μm. The parallel chip design was generated by arranging multiple devices side by side. The microfluidic chip was fabricated using standard photolithography and soft lithography procedures. The negative photoresist SU8-3025 (MicroChem) was used to fabricate patterns on a silicon wafer. The silicon wafer was then silanized using trimethylchlorosilane (Thermo Scientific) for 30 min to facilitate PDMS mold release. PDMS prepolymer (10A:1B, Sylgard 184 silicone elastomer kit, Dow Corning) was poured onto the silicon wafer and cured at 80°C for 1 hour. Holes were then punched in the PDMS for the inlets and outlets, and oxygen plasma treatment was used to chemically bond the PDMS mold to a glass slide.
Trimethylchlorosilane
Manufactured by Thermo Fisher Scientific
Sourced in United States
Trimethylchlorosilane is a chemical compound with the formula (CH3)3SiCl. It is a colorless, volatile liquid commonly used as a reagent in organic synthesis and for the preparation of other silicon-containing compounds.
Automatically generated - may contain errors
Lab products found in correlation
Su 8 3025, by MicroChem (3 mentions)
Autocad, by Autodesk (2 mentions)
Pdms prepolymer, by Dow (2 mentions)
Pyridine, by Merck Group (2 mentions)
C18 0methyl ester, by Thermo Fisher Scientific (2 mentions)
Formic acid, by Merck Group (2 mentions)
Silylation grade pyridine, by Avantor (2 mentions)
Sylgard 184 silicone elastomer kit, by Dow (1 mentions)
Acetonitrile of hplc grade, by Thermo Fisher Scientific (1 mentions)
Citric acid, by Merck Group (1 mentions)
Catechin, by Merck Group (1 mentions)
Hexane, by Avantor (1 mentions)
Phenylacetic acid, by Merck Group (1 mentions)
Benzoic acid, by Merck Group (1 mentions)
Acetonitrile, by Thermo Fisher Scientific (1 mentions)
Sucrose, by Merck Group (1 mentions)
Acetic acid, by Thermo Fisher Scientific (1 mentions)
Ethyl acetate, by Avantor (1 mentions)
Dichloromethane, by Avantor (1 mentions)
Epicatechin, by Merck Group (1 mentions)
16 protocols using trimethylchlorosilane
1
Microfluidic Chip Fabrication Using Photolithography
2
Analytical Standards for Phenolic Compounds
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Acetonitrile of HPLC grade, acetic acid and N,O-bis(trimethylsilyl)trifluoroacetamide + 10% trimethylchlorosilane (BSTFA + 10% TMCS) were from Fischer Scientific (Pittsburgh, PA, USA). 5-O-caffeoylquinic acid, (+)-catechin, (−)-epicatechin, sucrose, glucose, fructose, citric acid, NaBH4, N-methylimidazole, acetic anhydride, benzoic acid, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 3-phenylpropionic acid, phenylacetic acid, 3-hydroxyphenylacetic acid, 3-(4-hydroxyphenyl)propionic acid, dl -3-phenyllactic acid, dl -3-(4-hydroxyphenyl)lactic acid, 5-phenylvaleric acid, 3,4-dihydroxyphenylacetic acid and 2,4,5-trimethoxycinnamic acid and toluene-α−thiol were obtained from Sigma-Aldrich (Saint Quentin Fallavier, France). Phloretin, p-coumaric acid, quercetin and cyanidin-3-O-galactoside were obtained from Extrasynthese (Lyon, France). Phloridzin was obtained from Fluka (Buchs, Switzerland). Malic acid was obtained from R-Biopharm (Darmstadt, Germany). D3-Methanol was from Acros Organics (Geel, Belgium). Acetonitrile was analytical grade and from Fisher Scientific (Fair Lawn, NJ, USA). Ethyl acetate, dichloromethane and hexane obtained from VWR International (Radnor, PA, USA).
3
Silylation Procedure for GC-MS Analysis
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All chemicals and standards were purchased from Sigma-Aldrich unless otherwise stated. N-Methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) and trimethylchlorosilane (TMCS) were purchased from Thermo Scientific. LC–MS grade Water (H2O) and Methanol (CH3OH) were purchased from Rathburn Chemicals Ltd. Chloroform (CHCl3) analytical grade was purchased from Fisher Chemicals.
4
Derivatization and GC-MS Analysis of Metabolites
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Dried samples were resuspended in 50 µl of pyridine containing 25 mg/ml of methoxyamine hydrochloride, incubated at 60°C for 45 min, vigorously vortexed for 30 s, sonicated for 10 min, and incubated for an additional 45 min at 60°C. Next, 50 µl of N‐methyl‐N‐trimethylsilyltrifluoroacetamide with 1% trimethylchlorosilane (Thermo Fisher Scientific) was added, and samples were vigorously vortexed for 30 s and incubated at 60°C for 30 min. Metabolites were detected using a Trace 1310 GC coupled to a Thermo ISQ mass spectrometer. Samples (1 µl) were injected at a 10:1 split ratio to a 30 m TG‐5MS column (Thermo Fisher Scientific; 0.25 mm id, 0.25 µm film thickness) with a 1.2 ml/min helium gas flow rate. The GC inlet was held at 285°C. The oven program started at 80°C for 30 s, followed by a ramp of 15°C/min to 330°C, and an 8 min hold. Masses between 50 and 650 m/z were scanned at 5 scans/s under electron impact ionization. Transfer line and ion source were held at 300°C and 260°C, respectively. Pooled quality control (QC) samples were injected after every six actual samples. The analytical sample order was randomized.
5
Metabolomic Analysis of Cecal Contents
6
Microfluidic Device for Single-Cell Trapping
7
Urinary NAA Quantification by GCMS
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Rats were housed in metabolic cages after physiological saline was orally administered at
2.5 ml/100 g (body weight). Urine samples from rats were collected 6 h later. The
N-acetyl-L-aspartate (NAA) level in urine was measured using
gas chromatography mass spectrometry (GCMS) as previously described [10 (link)]. Briefly, 25 µl of urine was mixed with 5
µl of 1 mg/ml 2-isopropylmalic acid (Sigma–Aldrich) as an internal
standard. The mixture was treated with urease to decompose and remove excess urea. After
centrifuging the samples, the collected supernatant was dried under an N2 gas
stream and resolved in N,O-bis (trimethylsilyl) trifluoroacetamide with
10% trimethylchlorosilane (Thermo Scientific, Tokyo, Japan). The GCMS analysis was
performed using a GCMS-QP2010 Plus system (Shimadzu Corporation, Kyoto, Japan) with a DB-5
column (Agilent Technologies, Santa Clara, CA, USA).
2.5 ml/100 g (body weight). Urine samples from rats were collected 6 h later. The
N-acetyl-L-aspartate (NAA) level in urine was measured using
gas chromatography mass spectrometry (GCMS) as previously described [10 (link)]. Briefly, 25 µl of urine was mixed with 5
µl of 1 mg/ml 2-isopropylmalic acid (Sigma–Aldrich) as an internal
standard. The mixture was treated with urease to decompose and remove excess urea. After
centrifuging the samples, the collected supernatant was dried under an N2 gas
stream and resolved in N,O-bis (trimethylsilyl) trifluoroacetamide with
10% trimethylchlorosilane (Thermo Scientific, Tokyo, Japan). The GCMS analysis was
performed using a GCMS-QP2010 Plus system (Shimadzu Corporation, Kyoto, Japan) with a DB-5
column (Agilent Technologies, Santa Clara, CA, USA).
8
Microfluidic Cluster-Chip for CTC Capture
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The microfluidic chip pattern is based on the design of the paper [27 (link)]. The Cluster-Chip captures CTC clusters by relying on the strength of cell–cell junctions as clusters flow at physiological speed through a set of triangular pillars. Three pillars make up the basic unit of the chip; two form a narrowing channel that funnels the cells into an opening, where the edge of the third pillar is positioned to bifurcate the laminar flow. As blood flows, single blood and tumor cells divert to one of the two streamlines at the bifurcation and pass through the 12 µm × 100 µm opening. In contrast, CTC clusters are held at the edge of the bifurcating pillar. The microfluidic device was fabricated according to standard photolithography and soft lithography procedures. The negative photoresist SU8-3025 (MicroChem, USA) pattern on the silicon wafer was fabricated with a photomask. The silicon wafer was then silanized with trimethylchlorosilane (Thermo Scientific, USA) to facilitate PDMS mold release PDMS prepolymer (Dow Corning, USA) was poured onto the silicon wafer and cured at 80°C for 1 h. Holes were punched in the PDMS, and oxygen plasma treatment was used to chemically bond the PDMS mold to a glass slide.
9
GC/MS Analysis of Cecal Metabolites
10
Metabolomic Profiling via HPLC-GC-MS
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High-performance liquid chromatography (HPLC)-grade methanol was obtained from Thermo Fisher Scientific (Hampton, NH, USA). HPLC-grade hexanes were obtained from Honeywell Burdick & Jackson (Muskegon, MI, USA). Myristic-d27 acid, methoxylamine hydrochloride, and pyridine were obtained from Sigma Aldrich (St. Louis, MO, USA). N,O-bis(trimethylsilyl) trifluoroacetamide containing 1% trimethylchlorosilane was obtained from Alfa Aesar (Ward Hill, MA, USA).
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