Design of reactive porous polymer supports for high throughput bioreactors: Poly(2-vinyl-4,4-dimethylazlactone-co-acrylamide-co-ethylene dimethacrylate) monoliths

Xie, Shaofeng; Švec, František; Fréchet, Jean M. J.

doi:10.1002/(sici)1097-0290(19990105)62:1<30::aid-bit4>3.0.co;2-e

Cited by 179 publications

(122 citation statements)

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“…Typically, the reaction time required for homogeneous solution tryptic digestions is tens of hours to achieve a sufficient population of peptide fragments for effective PMF because the enzyme-to-substrate ratio must be kept low to avoid interferences from autodigested trypsin [26]. To overcome this problem, enzymeimmobilization on solid supports for solid-phase bioreactors has been introduced [26,[31][32][33]. Many of these solid-phase bioreactors use immobilization of proteolytic enzymes through covalent attachment to supports or encapsulation within gel matrices.…”

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confidence: 99%

“…Functionalized 3-D support networks can also be made in situ [31][32][33][34], which overcomes the difficulty of packing beads into the microchannels. Typically, these in situ methods use a polymerization reaction of a monomer solution inside a channel to modify the support.…”

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“…Then, trypsin solution is infused into the bioreactor for about 24 h for complete immobilization. Peterson et al [31] developed a porous organic polymer monolithic bioreactor as in situ approach that allowed trypsin to be immobilized covalently within the monolith using the azlactone functional groups of the monolith [32]. Kato et al [33] reported a monolithic photopolymerized sol-gel column that was used to immobilize pepsin within a fused silica capillary for integration of protein digestion and identification by CE/MS.…”

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Development of an automated digestion and droplet deposition microfluidic chip for MALDI-TOF MS

Lee

Musyimi

Soper

et al. 2008

J. Am. Soc. Mass Spectrom.

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An automated proteolytic digestion bioreactor and droplet deposition system was constructed with a plastic microfluidic device for off-line interfacing to matrix assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS). The microfluidic chips were fabricated in poly(methyl methacrylate) (PMMA), using a micromilling machine and incorporated a bioreactor, which was 100 ILm wide, 100 ILm deep, and possessed a 4 cm effective channel length (400 nL volume). The chip was operated by pressure-driven flow and mounted on a robotic fraction collector system. The PMMA bioreactor contained surface immobilized trypsin, which was covalently attached to the UV-modified PMMA surface using coupling reagents N-(3-dimethylaminopropyl)-N'-ethy1carbodiimide hydrochloride (EDC) and hydroxysulfosuccinimide (sulfo-NHS). The digested peptides were mixed with a MALDI matrix on-chip and deposited as discrete spots on MALDI targets. The bioreactor provided efficient digestion of a test protein, cytochrome c, at a flow rate of 1 ILL/min, producing a reaction time of -24 s to give adequate sequence coverage for protein identification. Other proteins were also evaluated using this solid-phase bioreactor. The efficiency of digestion was evaluated by monitoring the sequence coverage, which was 64%, 35%, 58%, and 47% for cytochrome c, Significant progress has been made toward the development of microchip-based technologies for proteomics through the integration of analytical processes into platforms that can provide rapid identification of proteins and the subsequent characterization of various post-translational modifications [1][2][3][4]. The small sample and reagent requirements, rapid analysis times, high throughput processing capabilities, and low operating costs are among the driving forces for the development of these systems [5][6][7][8]. Different microfluidic devices have been applied to specific aspects of protein processing, in particular, protein purification and separation, protein digestion, and protein identification by mass spectrometry [9].There have been a number of approaches to on-line and off-line matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) integrated to chipbased devices [10][11][12][13][14][15][16][17][18]. The primary advantage of the MALDI approach compared with electrospray ionization (ESI) when coupling to microfluidic chips is the potential for multiplexing [19]. The directional control Address reprint requests to Dr. Kermit K. Murray, Department of Chemistry, Louisiana State University, 232 Choppin Hall, Baton Rouge, LA 70802, USA. E-mail: kkmurray@!su.edu of the ESI spray can be difficult with a high-density array of spray tips. Furthermore, microfluidic chip interfaces to ESI can suffer from stability problems when sprays are started or stopped [20,21]. This limits the speed of moving from one sample to another and therefore, limits throughput.With recent advances in analytical methods for proteomics, attention has been directed toward development of effic...

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Development of an automated digestion and droplet deposition microfluidic chip for MALDI-TOF MS

Lee

Musyimi

Soper

et al. 2008

J. Am. Soc. Mass Spectrom.

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“…Azlactone readily reacts with nucleophiles such as amines and thiols at room temperature to form amide bonds. 182 Peterson et al reported a microfluidic protein digestion device using a photopatterned polymer monolith where trypsin was immobilized using azlactone. 61 Glass chips were silanized with 3-(trimethosysilyl)propyl methacrylate, leaving the methacryloyl group to covalently link polymer monolith to the glass substrate.…”

Section: Amine-azlactonementioning

confidence: 99%

Protein immobilization techniques for microfluidic assays

2013

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Microfluidic systems have shown unequivocal performance improvements over conventional bench-top assays across a range of performance metrics. For example, specific advances have been made in reagent consumption, throughput, integration of multiple assay steps, assay automation, and multiplexing capability. For heterogeneous systems, controlled immobilization of reactants is essential for reliable, sensitive detection of analytes. In most cases, protein immobilization densities are maximized, while native activity and conformation are maintained. Immobilization methods and chemistries vary significantly depending on immobilization surface, protein properties, and specific assay goals. In this review, we present trade-offs considerations for common immobilization surface materials. We overview immobilization methods and chemistries, and discuss studies exemplar of key approaches—here with a specific emphasis on immunoassays and enzymatic reactors. Recent “smart immobilization” methods including the use of light, electrochemical, thermal, and chemical stimuli to attach and detach proteins on demand with precise spatial control are highlighted. Spatially encoded protein immobilization using DNA hybridization for multiplexed assays and reversible protein immobilization surfaces for repeatable assay are introduced as immobilization methods. We also describe multifunctional surface coatings that can perform tasks that were, until recently, relegated to multiple functional coatings. We consider the microfluidics literature from 1997 to present and close with a perspective on future approaches to protein immobilization.

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“…[16 -19] In terms of applications, these new separation media, usually referred to as monolithic columns or rigid rods [20] have already been used successfully in standard HPLC and micro-separation techniques, [9, 21 -26] CEC as well as in solid phase extraction (SPE), [27] and more recently, as bioreactors. [28,29] Generally speaking, the term monolith applies to any unibody structure composed of interconnected repeating cells or channels. Such media may either be metallic or prepared from inorganic mixtures, e.g., by a sintering process to form ceramics, or from organic compounds, e.g., by a crosslinking polymerization.…”

Section: The Concept Of Monolithic Mediamentioning

confidence: 99%

Metathesis-Based Monolithic Supports: Synthesis, Functionalization and Applications

Buchmeiser

2001

Macromol. Rapid Commun.

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Design of reactive porous polymer supports for high throughput bioreactors: Poly(2-vinyl-4,4-dimethylazlactone-co-acrylamide-co-ethylene dimethacrylate) monoliths

Cited by 179 publications

References 14 publications

Development of an automated digestion and droplet deposition microfluidic chip for MALDI-TOF MS

Development of an automated digestion and droplet deposition microfluidic chip for MALDI-TOF MS

Protein immobilization techniques for microfluidic assays

Metathesis-Based Monolithic Supports: Synthesis, Functionalization and Applications

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