Immobilization of the restriction enzymesHaeIII andHindIII on porous silica particles via a glutaraldehyde linkage for the micro-digestion of dsDNA with analysis by capillary electrophoresis

In order to automate the optimization of complex biochemical and molecular biology reactions, we developed a Sequential Injection Analysis (SIA) device and combined this with a Design of Experiment (DOE) algorithm. This combination of hardware and software automatically explores the parameter space of the reaction and provides continuous feedback for optimizing reaction conditions. As an example, we optimized the endonuclease digest of a fluorogenic substrate, and showed that the optimized reaction conditions also applied to the digest of the substrate outside of the device, and to the digest of a plasmid. The sequential technique quickly arrived at optimized reaction conditions with less reagent use than a batch process (such as a fluid handling robot exploring multiple reaction conditions in parallel) would have. The device and method should now be amenable to much more complex molecular biology reactions whose variable spaces are correspondingly larger.

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“…That said, it has been noted by Davidson et. al 21. that the HaeIII enzyme shows an increased rate at higher DTT concentrations.…”

Section: Resultsmentioning

confidence: 95%

Sequential Injection Analysis for Optimization of Molecular Biology Reactions

Allen

Ellington

2011

Anal. Chem.

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“…At higher volumes of PEG-DM, the reaction solution was biphasic, owing to the decreased solubility of PEG-DM in toluene. It has been reported that modified hydrophilic surfaces can effectively reduce surface-induced denaturation of trypsin as well as reduce attractive protein−surface interactions observed with hydrophobic surfaces. − …”

Section: Resultsmentioning

confidence: 99%

Enhanced Proteolytic Activity of Covalently Bound Enzymes in Photopolymerized Sol Gel

2005

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Trypsin is covalently linked to a photopolymerized sol-gel monolith modified by incorporating poly(ethylene glycol) (PSG-PEG) for on-column digestion of N(alpha)-benzoyl-l-arginine ethyl ester (BAEE) and two peptides, neurotensin and insulin chain B. The coupling of the enzyme to the monolith is via room-temperature Schiff chemistry in which an alkoxysilane reagent (linker) with an aldehyde functional group links to an inactive amine on trypsin to form an imine bond. The proteolytic activity of the immobilized trypsin was measured by monitoring the formation of N alpha-benzoyl-L-arginine (BA), the digestion product of BAEE. The BA is separated from BAEE by capillary electrophoresis and detected downstream (18.5 cm from the microreactor) by absorption (254 nm). Using the Bradford assay, we determined that 97 ng of trypsin is bound to the 1-cm microreactor located at the entrance of capillary column. The bioactivity of the trypsin-PSG-PEG microreactor at 20 degrees C for the digestion of BAEE was found to be 2270 units/mg of immobilized trypsin. The bioactivity of trypsin bound to the capillary wall in the open segment upstream from the monolith was 332 units/mg of immobilized trypsin under the same conditions. In contrast, the activity of free trypsin could not be observed for the digestion of BAEE at 20 degrees C after 16 h of incubation time.

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“…As such, a diverse range of surface modification chemistries can be used for polymers to generate functional surfaces appropriate for the intended application, 18,19,24 which can consist of the surface immobilization of biological agents for recognition (nucleic acid probes, antibodies, etc. ), [25][26][27][28][29][30][31][32] formation of biocompatible surfaces (i.e., surface wettability), [33][34][35][36][37] immobilization of catalytic enzymes for solidphase bioreactors, 38,39 or solid-phase molecular extractions. [40][41][42] In addition, simple surface modification protocols can be used to generate functional groups through the use of ultraviolet (UV)-activation, [43][44][45] plasma oxidation, [46][47][48][49][50][51] reactive ion beams, 52 microwave-oven generated plasmas, 53 atom-transfer radical polymerizations, 54 and layer-by-layer techniques.…”

Section: Surface Modification Of Polymersmentioning

confidence: 99%

“…In either case, the selection element or enzyme are either covalently or noncovalently attached to a solid-support and the targets or substrates are solution-borne and can be driven through the reactor bed either hydrodynamically or electrokinetically. There are several advantages associated with solid-phase reactors as opposed to their homogeneous (solution) counterparts: (1) reuse of the immobilized reagent for subsequent analysis; 38,250,251 (2) enhanced stability and activity of the reagent when immobilized to a solid-support; [252][253][254] and (3) simplified on-line processing of the sample in fluidic systems as well as easier separation of the reaction products from the catalytic enzyme or removal of the interfering components from the selected target.…”

Section: Solid-phase Reactorsmentioning

confidence: 99%

“…), 25–32 formation of biocompatible surfaces ( i.e. , surface wettability), 33–37 immobilization of catalytic enzymes for solid-phase bioreactors, 38,39 or solid-phase molecular extractions. 40–42 In addition, simple surface modification protocols can be used to generate functional groups through the use of ultraviolet (UV)-activation, 43–45 plasma oxidation, 46–51 reactive ion beams, 52 microwave-oven generated plasmas, 53 atom-transfer radical polymerizations, 54 and layer-by-layer techniques.…”

Section: Introductionmentioning

confidence: 99%

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Flexible fabrication and applications of polymer nanochannels and nanoslits

et al. 2011

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Fluidic devices that employ nanoscale structures (<100 nm in one or two dimensions, slits or channels, respectively) are generating great interest due to the unique properties afforded by this size domain compared to their micro-scale counterparts. Examples of interesting nanoscale phenomena include the ability to preconcentrate ionic species at extremely high levels due to ion selective migration, unique molecular separation modalities, confined environments to allow biopolymer stretching and elongation and solid-phase bioreactions that are not constrained by mass transport artifacts. Indeed, many examples in the literature have demonstrated these unique opportunities, although predominately using glass, fused silica or silicon as the substrate material. Polymer microfluidics has established itself as an alternative to glass, fused silica, or silicon-based fluidic devices. The primary advantages arising from the use of polymers are the diverse fabrication protocols that can be used to produce the desired structures, the extensive array of physiochemical properties associated with different polymeric materials, and the simple and robust modification strategies that can be employed to alter the substrate's surface chemistry. However, while the strengths of polymer microfluidics is currently being realized, the evolution of polymer-based nanofluidics has only recently been reported. In this critical review, the opportunities afforded by polymer-based nanofluidics will be discussed using both elastomeric and thermoplastic materials. In particular, various fabrication modalities will be discussed along with the nanometre size domains that they can achieve for both elastomer and thermoplastic materials. Different polymer substrates that can be used for nanofluidics will be presented along with comparisons to inorganic nanodevices and the consequences of material differences on the fabrication and operation of nanofluidic devices (257 references).

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Immobilization of the restriction enzymesHaeIII andHindIII on porous silica particles via a glutaraldehyde linkage for the micro-digestion of dsDNA with analysis by capillary electrophoresis

Cited by 4 publications

References 27 publications

Sequential Injection Analysis for Optimization of Molecular Biology Reactions

Sequential Injection Analysis for Optimization of Molecular Biology Reactions

Enhanced Proteolytic Activity of Covalently Bound Enzymes in Photopolymerized Sol Gel

Flexible fabrication and applications of polymer nanochannels and nanoslits

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