Insulator Nanostructure Desorption Ionization Mass Spectrometry

6 days ago - Abstract Image ... metabolites, INDI-MS mass spectra of 10 amol verapamil, observed INDI-MS m/z ... ac8b01989_si_001.pdf (1.31 MB) ... ar...
4 downloads 0 Views 6MB Size
Download PDF
Subscriber access provided by UNIV OF DURHAM

Letter

Insulator Nanostructure Desorption Ionization Mass Spectrometry Todd A. Duncombe, Markus de Raad, Benjamin P. Bowen, Anup K Singh, and Trent R. Northen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01989 • Publication Date (Web): 31 Jul 2018 Downloaded from http://pubs.acs.org on August 3, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Insulator Nanostructure Desorption Ionization Mass Spectrometry Todd A. Duncombe1,3,†, Markus De Raad4, Benjamin P. Bowen2,4, Anup K. Singh1,3, & Trent Northen1,2,4,* 1

DOE Joint BioEnergy Institute, 5885 Hollis Street, Emeryville, California 94608, United States. Joint Genome Institute, Department of Energy, 2800 Mitchell Drive, Walnut Creek, California 94598, United States. 3 Sandia National Laboratories, Livermore, California 94550, USA 4Environmental Genomics and Systems Biology, Biosciences, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, California 94720, United States. 2

ABSTRACT: Surface-Assisted Laser Desorption Ionization (SALDI) is an approach for gas phase ion generation for mass spectrometry using laser excitation on typically conductive or semi-conductive nanostructures. Here, we introduce Insulator Nanostructure Desorption Ionization Mass Spectrometry (INDI-MS), a nanostructured polymer substrate for SALDI-MS analysis of small molecules and peptides. INDI-MS surfaces are produced through the self-assembly of a perfluoroalkyl silsequioxane nanostructures in a single chemical vapor deposition silanization-step. We find that surfaces formed from the perfluorooctyltrichlorosilane monomer assemble semielliptical-features with a 10 nm height, diameters between 10 to 50 nm, and have attomole-femtomole sensitivities for selected analytes. Surfaces prepared with silanes that either lack the trichloro or perfluoro groups, lack sensitivity. Further, we demonstrate that hydrophobic INDI regions can be micropatterned onto hydrophilic surfaces to perform on-chip self-desalting in an array format. Laser desorption ionization mass spectrometry (LDI-MS) has long been used to analyze a broad range of compounds, ranging from metabolites to biopolymers. Surface-Assisted Laser Desorption Ionization (SALDI) is an approach of gas phase ion generation that uses laser excitation of surfaces for mass spectrometry (MS).1,2 SALDI surfaces typically utilize nanostructures prepared from a wide variety of conductive and semi-conductive materials, including nanostructured silicon,3 carbon nanotubes, and metal nanoparticles.4,5 Many SALDI techniques display high sensitivity due to a low-chemical background. For example, Nanostructure-Initiator Mass Spectrometry (NIMS) combines a SALDI surface with a perfluorous liquid ‘initiator’ to facilitate desorption and has been used for the analyses of a broad range of small molecules, metabolites, lipids, and peptides with limits of detection down to the yoctomole level.6–8 Despite performance advantages, the challenges associated with the fabrication of SALDI surfaces limits their use to labs specialized in nanofabrication. For example, NIMS surfaces are typically generated by anodic hydrofluoric acid etching of silicon, which requires special training and equipment. Recently, our group developed a method of generating black silicon NIMS using plasma etching.9 While this was an important advance because it eliminated the wet hydrofluoric acid etching step, it still required highly specialized equipment typically only found in clean-rooms. An emerging alternative to solid-state materials is the use of polymers. In recent years, organic electronics have been widely adopted for optoelectronics.10 This is in large part due to their relatively low cost, ease of fabrication, and their mechanical and chemical stability. Organic electronics regularly use simple and high-throughput fabrication procedures including chemical vapor deposition (CVD), sputtering, and spin coating.11 Organically modified silica gels, also referred to as organic silsequiox-

anes,12 can be similarly deposited atop silica or silicon substrates via a simple condensation reaction to achieve various chemical properties, such as enhanced photochemical activities,13 or to modify surface energy.14 When the silica-gel monomer contains a tri-valent reaction site, such as an alkyltrichlorosilane, they can under certain conditions self-assemble into nanobead-structures during polymerization.15 In this work we describe a high-performing perfluoroalkyl silsequioxane16 coating for SALDI-MS. Our approach, coined Insulator Nanostructure Desorption Ionization Mass Spectrometry (INDI-MS) to differentiate this polymer-based approach from existing metal/semiconductor SALDI surfaces, utilizes the low-cost and simplicity of organic electronics fabrication to generate NIMS-like SALDI surfaces. To gain insights into the surfaces we compare materials and find that the trichlorosilane and perfluorous moieties to be important components. We show that INDI-MS can be integrated with photolithography and exhibit a micropattern for sample self-desalting in an array format.

Figure 1: Insulator Nanostructure Desorption Ionization Mass Spectrometry (INDI-MS) utilizes self-assembled perfluoroalkyl nanostructures as a LDI-MS platform. (A) INDI substrates are

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

generated in 20 min using (I) CVD of FOTS which (II) reacts with silicon oxide surface and to itself to form (III) nanostructures with a siloxane backbone (black) decorated with fluorocarbon side groups (yellow).15 (B) AFM and (C) SEM demonstrate the 10 nm to 50 nm diameter semielliptical nanostructures spaced by ~100 nm on INDI surfaces. (D) LDI-MS is performed directly on the INDI surface to analyze adsorbed molecules. (E) INDI-MS mass spectra of a sample containing 250 fmols of dextromethorphan, verapamil, palmitoyl carnitine, 2.5 pmol mastoparan, and background (‘*’).

Materials and Methods SALDI Surface Fabrication. In this work, wet-etched and black silicon NIMS surfaces are prepared as described previously.6,9 INDI-MS substrate fabrication is performed in 25 minutes using readily available materials and equipment: a plasma cleaner, hot pate, dessicator, vacuum, and perfluorooctyltrichlorosilane (FOTS, Sigma Aldrich, St Louis, MO). The simple protocol (see Supporting Information Protocol S-1 for a step-by-step protocol) was adapted from a previous publication.15 New silicon wafers (n-type prime-grade, University Wafer, South Boston, MA) are treated in an oxygen plasma chamber for 5 min at 600 mTorr. Then, they are immediately placed in the vacuum chamber with 200 µL FOTS, followed by the application of house vacuum (100 Torr) for 5 min. Then, the chamber is sealed to maintain the vacuum and transferred onto a 150 °C hot plate for 10 min. Once the wafer has cooled, the INDI-MS surfaces are ready to use. INDI-MS substrates are one-time use. Alternative silanization-based coatings of octyltrichlorosilane (OTS, Sigma Aldrich) and perfluorooctydimethylchlorosilane (FOCS, Alfa Aesar, MA) were fabricated with the same protocol and used as controls. Micropatterned INDI substrate fabrication is described in Supporting Information Protocol S-2, it incorporates photolithography and metal etching to generate INDI-MS micropatterns with their locations indicated by aluminum traces. Sample Preparation, Acoustic printing, and MS Measurements. Unless otherwise noted, INDI-MS measurements were conducted as follows. Aqueous samples containing 0.2% formic acid were spotted with a volume of 10 nL using an EDC ATS-100 acoustic transfer system (Fremont, CA). Individual mass spectra and Mass Spectrometry Imaging (MSI) was performed using an AB Sciex 5800 MALDI TOF/TOF mass spectrometer (Foster City, CA) in positive mode. Laser intensity ranged between 4500 and 6000 arbitrary units and was optimized for each substrate type. For quantitation, MSI images were collected using surface rasterization at a 75 µm step size, m/z range of 50 to 2000, 20 laser shots per spectrum, and then analyzed using the previously published OpenMSI and OpenMSI Array Analysis Toolkit.17,18 Analytes dextromethorphan, palmitoyl carnitine, and arginine were purchase from Sigma Aldrich. STAL-2 and Mastoparan were purchased from AnaSpec (Freemont, CA). Secondary Metabolite Screening. INDI-MS analyzed a 288 secondary metabolite library from Enzo Life Science (Lausen, Switzerland). The 0.1 mg/mL samples were diluted 1:8 in a 20% methanol solution, four replicates were acoustically printed on an INDI substrate, and then INDI-MS imaging was performed (see Supporting Information Method S-1). NIMS-based Enzyme Screen. A mixture of 1 mM substrates developed for NIMS enzyme assays (cellobiose, mannobiose, and xylobiose derivatized with perfluoronated ‘tags’)19 were mixed with Cellic CTec2 and HTec2 cellulases and hem-

Page 2 of 5

icellulases blends (Novozymes, Bagsvaerd, Denmark) to monitor enzyme activity. Reactions were incubated at 55° C and 2 µL samples were taken at 0, 3, 10 and 30 minutes, then quenched in 2 uL of ice-cold methanol, and analyzed with INDI-MS imaging (see Supporting Information Method S-2). Surface Characterization. Atomic force microscopy (AFM) was performed in tapping-mode on an Asylum MFP-3D (Santa Barbara, CA). A Zeiss Gemini Ultra-55 (Jena, Germany) scanning electron microscopy (SEM) was operated at 1 kV and images were captured immediately to minimize charging. Results and Discussion INDI substrates are generated with a common fabrication technique used for functionalizing oxide surfaces, the CVD of alkyltrichlorosilanes. In the presence of water vapor and a silicon wafer, depicted in Fig. 1A, the three reaction sites of trichlorosilane rapidly form self- and surface-siloxane bonds through a hydrolytic condensation. As the CVD proceeds, a step-growth polymerization generates silsesquioxane nanoparticles anchored covalently to the silicon surface. A previous publication demonstrated that the size of the self-assembled nanostructures is tunable by modifying the reaction time.15 In Fig. 1B & 1C, AFM and SEM micrographs display the nanostructured INDI substrate, respectively. Both measurements confirmed the generation of semielliptical nanostructures during the CVD self-assembly process. The approximate nanostructures had a circular cross-section of 50 nm, a height of 10 nm, and a nanostructure to nanostructure spacing ranging between 75 nm and 200 nm. INDI surfaces can be used immediately after fabrication for the LDI-MS analysis of adsorbed small-molecules – as depicted in Fig. 1D & 1E. INDI-MS analysis for the drug verapamil, as shown in Fig. S-1, was detected at 10 amol with a S/N of ~200. While not as sensitive as conventional wet etched NIMS surfaces this is comparable to black silicon NIMS surfaces and useful for many applications.9

Figure 2: INDI-MS comparison to alternative siloxane Si-coatings and previously published NIMS platforms. (A) AFM confirmed that the molecules with trivalent reactant-sites, FOTS and OTS, form similar nanostructures, while the monovalent FOCS formed a monolayer with ~1 nm features. (B) LDI-MS for FOTS, OTS, FOCS and plain Si surfaces were compared for the detection of 10-13 mol dextromethorphan [Dex.]. (C) LDI-MS of INDI-MS, wet-etched NIMS, and black silicon NIMS substrates were compared. Analytes: 10-14 mol Dex., 10-13 mol palmitoyl carnitine [Pal.], 10-14 mol verapamil [Verap.], 10-13 mol STAL-2, and 10-13 mol mastoparan [Masto.]. [STD, n = 5]

To gain insights into the physical features responsible for INDI-MS performance, we fabricated alternative insulator coatings OTS and FOCS as controls. The trichlorosilane moiety featured on the OTS molecule maintains the trivalent self- and ox-

ACS Paragon Plus Environment

Page 3 of 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

ide-reactivity for forming nanostructures but lacks perfluorination of FOTS. In contrast, FOCS has an identical perfluorination as FOTS, but due to its single chlorosilane reaction site, chain-polymerization and thus silsesquioxane formation is not possible under these conditions. When interrogated with AFM, as shown in Fig. 2A, we find that OTS coated surfaces formed semielliptical nanostructures similar to FOTS-based INDI surfaces, while FOCS coated surfaces formed a monolayer with approximately a 1 nm thick film. The MS performance was compared between FOTS, OTS, and FOCS coatings, and plain Si, for the detection of 100 fmols of dextromethorphan in Fig. 2B. FOCS and OTS coatings had minimal MS-activity, only 2.3 and 1.8 times that of untreated Si wafers. In contrast, a >100-times MS-enhancement was observed for the FOTS coated substrate, saturating the MS detector. Thus, insulator coatings that are perfluorinated but not nanostructured, or vice versa, do not have the same high performance LDI-MS functionality as those that are both perfluorinated and naostructured—suggesting that this combination of attributes is important contributors to INDI performance. In addition, we find that INDI-MS does seem to perform well in negative instrument polarity, similar to conventional NIMS performed using fluorocarbon-based initiators.7 To assess performance of INDI-MS against conventional wet-etched NIMS and black silicon NIMS, chemically diverse small molecules were individually acoustically printed onto each substrate. The molecules included peptides mastoparan (observed H+ monoisotopic ion: 1478.95 m/z) and STAL-2 (748.42 m/z), the lipid palmitoyl carnitine (400.36 m/z), small drug molecules dextromethoprhan (272.20 m/z) and verapamil (455.30 m/z), and the amino acid arginine (175.11 m/z) as shown in Fig. 2C (see Supporting Information Table S-1 for expected and observed m/z values). For all molecules tested, both INDI-MS and NIMS had significantly better performance than black silicon NIMS. For the smaller molecules dextromethorphan and palmitoyl carnitine, INDI-MS had comparable signal to NIMS within the 10 to 100 fmol range. However, for larger molecules, INDI-MS did not perform as well as NIMS. At 100 fmols of STAL-2, the INDI-MS signal was ~50% of the NIMS signal. Despite this, the INDI-MS sensitivity is sufficient for many applications focused on small molecules (10 was determined to be 100 amol for dextromethorphan and verapamil, 1 fmol for STAL-2, mastoparan and palmitoyl carnitine, and 1 pmol for arginine. When using manual signal accumulation, as opposed to MSI, the limit of detection could be improved by approximately 10-fold, as shown in Fig. S-1 for measuring 10 amols of verpamil. The mass spectra of the ions at an abundance where the isotopic peaks are clearly observed is shown in Fig. 3B. To broadly examine molecules detectable on INDI-MS we analyzed a 288 compound secondary metabolite library from Enzo life sciences. We find that 37% of metabolites were detected in positive instrument polarity, comparable to a similar screen conducted black silicon NIMS.9 Detected metabolites are listed in Supporting Information Table S-2. Fig. 4A is a representative total ion image of a portion of the printed metabolite array and Fig 4B shows the average mass spectra for three metabolites. Similar to black silicon NIMS, most detectable compounds (84%) contained at least 1 nitrogen atom. Enzyme characterization has proven to be a major application for NIMS. To evaluate INDI-MS for this application we used established NIMS glycosyl hydrolase probes to compare the performance of two commercial cellulases and hemicellulases enzyme cocktails.19 Mixtures containing 1 mM NIMS substrates (disaccharides modified with a fluorous tag that retains them on the NIMS surface) were incubated with these enzyme cocktails and samples were periodically collected. CTec2 exhibits higher activity than HTec2, including the rapid hydrolysis for cellobiose (Fig. 4C & 4D).

Figure 4: INDI-MS Screening. (A) A representative total ion content INDI-MS image for the screening 288 secondary metabolites. (B) The average mass spectra of star-indicated metabolites in A. (C and D) plots of the results of the INDI enzyme screen for the Cellic CTec2 vs. HTec2 degradation of substrates cellobiose, xylobiose, and mannobiose at time points 0, 2, 10, and 30 min. [STD, n=4] While discovering the mechanism(s) underlying INDI-MS is beyond the scope of this work we attribute the absorption of light to plasmonic effects previously described for dielectric nanoparticle arrays.20 We speculate that laser absorption heats the

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

perfluorus silsesquioxane nanoparticles driving nanostructure rearrangement/ablation that releases adsorbed analyte, possibly along with adsorbed/trapped unpolymerized silane, analogous to the initiator molecules in NIMS.6 We have not observed significant in-source fragmentation, suggesting that this is a relatively ‘soft’ SALDI technique like NIMS. A common challenge for LDI-MS when analyzing complex samples is the signal suppression from salts. Previous researchers addressed this by incorporating a pull-down chemistry for molecules of interest followed by a washing step.21 Alternatively, self-desalting substrates have been established by realizing hydrophilic/hydrophobic surface patterns to differentially fractionate salts and hydrophobic molecules as a sample dries.22,23 INDI surfaces would be expected to be compatible with conventional photolithographic micropatterning since the FOTSbased INDI coating is covalently attached to the substrate and is produced through a deposition process.24 Following a simple protocol - Supporting Information Protocol S-2 – we generated self-desalting INDI-MS arrays. The self-desalting design, shown in Fig. 5A, consisted of a 4 mm diameter spot comprised of outer INDI ring enclosing angularly-periodic patterns of hydrophobic-MS-active INDI regions and hydrophilic-MSinactive silicon regions. Self-desalting, depicted in Fig. 5B, occurs passively as a sample dries with hydrophobic molecules of interest assembling on the INDI regions and salts depositing on the silicon. Desalting with this approach does not work for hydrophilic compounds as they colocalize with the salt in the silicon region. To visualize the arrays and test analyte adsorption, surfaces were soaked in a 10 µM solution of dextromethorphan for 30 min, these were then blown dry with nitrogen and MSimaged (Fig. 5C). Dextromethorphan was detected on INDIMS regions (red) but not from the silicon (black) in Fig. 5D.

Page 4 of 5

top panel photograph shows the fractionation of salts into the designated silicon regions and aluminum traces designate the location of the spot. The middle panel displays the INDI-MSI for STAL-2. In the bottom panel the composite images is displayed.

Self-desalting was demonstrated in Fig. 5E, by applying 6 µL samples containing 1 pmol STAL-2 with different Phosphate Buffer Saline (PBS) concentrations, ranging from 0 to 600 mM in total salt, corresponding to 0X, 0.5X, 1X, 2X, and 4X PBS buffers.25 The photograph of the desalted samples shown in the top panel of Fig. 5E highlights the localization of salt in the hydrophilic silicon regions. MS imaging revealed that in all samples the STAL-2 could only be detected in the INDI regions (Fig. 5E, middle panel; overlay image Fig. 5E bottom panel). For less than or equal to 1X PBS nearly the entire INDIMS pattern is observed. At 2X and 4X PBS, large crystals obstructed a portion of the micropatterned INDIs region. These results suggest that self-desalting INDI-MS could be adopted as a facile approach to fractionate samples containing 150 mM total salt (1X PBS) or less. In the future, micropattern design will be optimized to improve performance and the array up-scaled to desalt thousands of samples in parallel. Further, as INDI-MS patterns are pre-defined, a LDI-MS acquisition protocol could analyze only the de-salted/INDI-MS regions rather than a full MSI raster, enabling high-throughput analysis. Conclusions INDI-MS substrates represent a new SALDI surface for the analysis of small molecules. INDI surfaces are composed of perfluoroalkyl silsesquioxane nanostructures produced using a one-step CVD process, making their production accessible to most laboratories. To explore integration of INDI with microfabrication, we patterned hydrophobic INDI regions on a hydrophilic surface to create wettability arrays for a self-desalting function. In the future, efforts will focus on modifying INDIMS for the detection of anions (e.g. by incorporating aminosilane moieties7), determining the mechanisms of INDI-MS, and incorporating this approach into microfluidic devices to investigate microbial interactions. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications Website. It contains Supplemental Protocols for fabrication, Figure S-1 displays INDI-MS detection of 10 amol verapamil, and Table S-1 compares INDI-MS m/z vs theoretical values.

AUTHOR INFORMATION Corresponding Author *Email: [email protected] (T.N.)

Present Addresses †ETH Zürich D-BSSE, Basel, Switzerland Figure 5: Micropatterened INDI-MS array for self-desalting. (A) Self-desalting spots consist of a hydrophobic / LDI-MS active region and a hydrophilic / LDI-MS inactive region. (B) As a sample dries hydrophobic molecules adsorb to the INDI region and salts deposit onto the bare silicon. (C) The MSI and (D) mass spectra of absorbed dextromethorphan demonstrates the INDI micropattern and LDI-MS activity. (E) Self-desalting was performed, on a different pattern than in A-D, for 1 pmol STAL-2 with total salt concentration ranging between 0 and 600 mM (0X to 4X PBS). The

AKNOWLEDGEMENTS This work is a collaboration by ENIGMA- Ecosystems and Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov), a Scientific Focus Area Program at Lawrence Berkeley National Laboratory, the DOE Joint BioEnergy Institute (http://www.jbei.org) under contract number DE-AC0205CH11231 supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research.

ACS Paragon Plus Environment

Page 5 of 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

REFERENCES

(13)

(1)

(14)

(2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12)

Picca, R. A.; Calvano, C. D.; Cioffi, N.; Palmisano, F. Nanomaterials 2017, 7 (4), 75–95. Stolee, J. a.; Walker, B. N.; Zorba, V.; Russo, R. E.; Vertes, A. Phys. Chem. Chem. Phys. 2012, 14 (24), 8453–8471. Go, E. P.; Uritboonthai, W.; Apon, J. V.; Trauger, S. A.; Nordstrom, A.; O’Maille, G.; Brittain, S. M.; Peters, E. C.; Siuzdak, G. J. Proteome Res. 2007, 6 (4), 1492–1499. Chiang, C.-K.; Chen, W.-T.; Chang, H.-T. Chem. Soc. Rev. 2011, 40 (3), 1269–1281. Law, K. P.; Larkin, J. R. Anal. Bioanal. Chem. 2011, 399 (8), 2597–2622. Woo, H.-K.; Northen, T. R.; Yanes, O.; Siuzdak, G. Nat. Protoc. 2008, 3 (8), 1341–1349. Amantonico, A.; Flamigni, L.; Glaus, R.; Zenobi, R. Metabolomics 2009, 5 (3), 346–353. Northen, T. R.; Yanes, O.; Northen, M. T.; Marrinucci, D.; Uritboonthai, W.; Apon, J.; Golledge, S. L.; Nordström, A.; Siuzdak, G. Nature 2007, 449 (7165), 1033–1036. Gao, J.; de Raad, M.; Bowen, B. P.; Zuckermann, R. N.; Northen, T. R. Anal. Chem. 2016, 88 (3), 1625–1630. Babudri, F.; Farinola, G. M.; Naso, F.; Ragni, R. Chem. Commun. 2007, 0, 1003–1022. Dimitrakopoulos, C. D.; Malenfant, P. R. L. Adv. Mater. 2002, 14 (2), 99–117. Baney, R. H.; Itoh, M.; Sakakibara, A.; Suzuki, T. Chem. Rev. 1995, 95 (5), 1409–1430.

(15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25)

Dash, S.; Mishra, S.; Patel, S.; Mishra, B. K. Adv. Colloid Interface Sci. 2008, 140 (2), 77–94. Owen, M. J.; Williams, D. E. J. Adhes. Sci. Technol. 1991, 5 (4), 307–320. Oyola-Reynoso, S.; Tevis, I. D.; Chen, J.; Chang, B. S.; Çinar, S.; Bloch, J.-F.; Thuo, M. M. J. Mater. Chem. A 2016, 4 (38), 14729– 14738. Ramirez, S.; Mabry, J. Handb. Fluoropolymer Sci. Technol. 2014, 545–563. Rübel, O.; Greiner, A.; Cholia, S.; Louie, K.; Bethel, E. W.; Northen, T. R.; Bowen, B. P. Anal. Chem. 2013, 85 (21), 10354– 10361. De Raad, M.; De Rond, T.; Rübel, O.; Keasling, J. D.; Northen, T. R.; Bowen, B. P. Anal. Chem. 2017, 89 (11), 5818–5823. Deng, K.; George, K. W.; Reindl, W.; Keasling, J. D.; Adams, P. D.; Lee, T. S.; Singh, A. K.; Northen, T. R. Rapid Commun. Mass Spectrom. 2012, 26 (6), 611–615. Zhang, L.; Ge, C.; Zhang, K.; Tian, C.; Fang, X.; Zhai, W.; Tao, L.; Li, Y.; Ran, G. J. Opt. 2016, 18 (12), 125002–125006. de Rond, T.; Peralta-Yahya, P.; Cheng, X.; Northen, T. R.; Keasling, J. D. Anal. Bioanal. Chem. 2013, 405 (14), 4969–4973. Zeng, Z.; Wang, Y.; Shi, S.; Wang, L.; Guo, X.; Lu, N. Anal. Chem. 2012, 84 (5), 2118–2123. Zeng, Z.; Wang, Y.; Guo, X.; Wang, L.; Lu, N. J. Am. Soc. Mass Spectrom. 2014, 25 (5), 895–898. Duncombe, T. A.; Parsons, J. F.; Bo, K. F.; Böhringer, K. F. Langmuir 2012, 28 (38), 13765–13770. Cold Spring Harb. Protoc. 2006, DOI: 10.1101/pdb.rec8247.

ACS Paragon Plus Environment