Scanning electron microscopy study of protein immobilized on SIO2 Sol-gel surfaces

Assis, O. B. G.

doi:10.1590/s0104-66322003000300014

Cited by 11 publications

(7 citation statements)

References 6 publications

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“…While, to the best of our knowledge, no detailed adsorption study is available for the Taq, a number of prior studies using other commonly used proteins such as lysozyme (Assis 2003;Beverung et al 1999;Haynes et al 1994;Luk et al 2000;Norde and Favier 1992;Prime and Whitesides 1993;Story et al 1991;Yoon and Garrell 2003), creatine phosphokinase (Pancera and Petri 2002), glucose 6-phosphate dehydrogenase (Pancera and Petri 2002), bovine serum albumin Norde and Favier 1992;Popat and Desai 2004;Sweryda-Krawiec et al 2004;Yoon and Garrell 2003), immunoglobulin (Vermeer et al 2001), and many other proteins/enzymes have invariably shown unique adsorption rates on different materials, including some of the materials/surface presented in this work. The differences in rate of adsorptions may be attributed to a number of inter-playing mechanisms commonly postulated in literature including: tendency of protein hydrophobic/hydrophilic chain to either align towards (or away) from the adsorbing surface (Koutsopoulos et al 2004;Orasanu-Gourlay and Bradley 2006;Pancera and Petri 2002), surface free-energy (i.e., charge) (Noinville et al 2002), electrostatic attraction/repulsion (Assis 2003;Haynes et al 1994;Yoon and Garrell 2003), thermodynamics Norde and Haynes 1995), unique interfacial tension between the protein and adsorbing surface (Beverung et al 1999), and relationship between protein penetration and steric hindrance from the structure of the protein and adsorbing material (Luk et al 2000;Moskovitz and Srebnik 2005;Sofia et al 1998). It is worth noting that many authors have suggested that the adsorption mechanisms themselves may not be fully understood Luk et al 2000;Sweryda-Krawiec et al 2004;…”

Section: Discussionmentioning

confidence: 99%

Characteristics and impact of Taq enzyme adsorption on surfaces in microfluidic devices

Prakash

Amrein

Kaler

2007

Microfluid Nanofluid

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We investigated the adsorption of Taq enzyme, using sessile droplets, on different microfluidic materials. In propagating adsorption materials, the contact angle (CA) of a sessile Taq droplet continually recedes and collapses due to adsorption. Contrastingly, in contained adsorption materials it exhibits an initial reduced CA due to an instantaneous adsorption, however remains timeinvariant. Spectrophotometer analysis on SU8, a propagating adsorption material, reveals a gradual loss of Taq from the droplet onto the surface during droplet collapsing, as opposed to a rapid saturated adsorption in Teflon, a contained adsorption material. AFM micrographs of the adsorbed surfaces suggest a network-like structure in SU8 and distinctly different pillar-like structures in Teflon. With this understanding, we have successfully applied a SU8-Teflon coating to impart a time-invariant contact angle with minimal loss of Taq in surface microfluidic devices.

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Section: Discussionmentioning

confidence: 99%

Characteristics and impact of Taq enzyme adsorption on surfaces in microfluidic devices

Prakash

Amrein

Kaler

2007

Microfluid Nanofluid

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“…Pure silica 607 1.80 0.37 Sol-gel-encapsulated CGTase 484 1.79 0.36 Figure 6 shows in addition the characteristic bands of pure silica, that is, 950 (Si-O-Si, axial deformation), 810 (Si-O-Si, axial deformation), and 600 cm −1 (Si-O-Si, angular deformation) [23,24]. The characteristic peaks for the hydroxyl group bonds can also be observed in the range of 3,400 cm −1 [23].…”

Section: Methodsmentioning

confidence: 99%

Methods and Supports for Immobilization and Stabilization of Cyclomaltodextrin Glucanotransferase from Thermoanaerobacter

Amud

Silva

Tardioli

et al. 2008

Appl Biochem Biotechnol

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Thermoanaerobacter cyclomaltodextrin glucanotransferase (CGTase) was immobilized using different supports and immobilization methods to study the effect on activity recovery. The enzyme covalently attached into glyoxyl-silica showed low activity recovery of 1.5%. The hydrophobic adsorption of the enzyme on Octadecyl-Sepabeads yielded also low activity recovery, 3.83%, and the enzyme could easily leak from the support at low ionic strength, although the immobilization yield was satisfactory, approximately 76%. The CGTase encapsulated in a sol-gel matrix gave an activity recovery of 6.94% and maximum cyclization activity at 60 degrees C, at pH 6.0. The half-time life at 60 degrees C, pH 6.0, in the presence of substrate was 100 min, which was lower than that of the free enzyme. The best activity recovery in this work (6.94%) is approximately five times smaller than that obtained previously using glyoxyl-agarose as support and covalent immobilization. Thus, the best support and method we tested so far for immobilization of CGTase is covalent attachment on glyoxyl-agarose.

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“…Recent studies by Sweryda-Krawiec et al (2004) and Prakash et al (2008) have demonstrated adsorption by both hydrophobic and hydrophilic substrates, discounting wettability as a standalone mechanism. Prakash et al (2008) lists a multitude of possible interactions, including hydrophobicity/hydrophilicity (Koutsopoulos et al 2004;Orasanu-Gourlay and Bradley 2006;Pancera and Petri 2002), surface free-energy (Noinville et al 2002), electrostatic attraction/repulsion (Assis 2003;Haynes et al 1994;Yoon and Garrell 2003), thermodynamics (Haynes and Norde 1995;Norde and Haynes 1995), unique interfacial tension between the protein and adsorbing surface (Beverung et al 1999), and relationship between protein penetration and steric hindrance from the structure of the protein and adsorbing substrate (Luk et al 2000;Moskovitz and Srebnik 2005;Sofia et al 1998). It is worth noting that many authors have suggested that the adsorption mechanisms themselves may not be fully understood (Haynes and Norde 1995;Luk et al 2000;Sweryda-Krawiec et al 2004;Yeo et al 2006).…”

Section: Introductionmentioning

confidence: 99%

Sensitive, microliter PCR with consensus degenerate primers for Epstein Barr virus amplification

Phaneuf

Pak

et al. 2012

Biomed Microdevices

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Sensitive identification of the etiology of viral diseases is key to implementing appropriate prevention and treatment. The gold standard for virus identification is the polymerase chain reaction (PCR), a technique that allows for highly specific and sensitive detection of pathogens by exponentially amplifying a specific region of DNA from as little as a single copy through thermocycling a biochemical cocktail. Today, molecular biology laboratories use commercial instruments that operate in 0.5-2 h/analysis using reaction volumes of 5-50 μL contained within polymer tubes or chambers. Towards reducing this volume and maintaining performance, we present a semi-quantitative, systematic experimental study of how PCR yield is affected by tube/chamber substrate, surface-area-to-volume ratio (SA:V), and passivation methods. We perform PCR experiments using traditional PCR tubes as well as using disposable polymer microchips with 1 μL reaction volumes thermocycled using water baths. We report the first oil encapsulation microfluidic PCR method without fluid flow and its application to the first microfluidic amplification of Epstein Barr virus using consensus degenerate primers, a powerful and broad PCR method to screen for both known and novel members of a viral family. The limit of detection is measured as 140 starting copies of DNA from a starting concentration of 3 × 10(5) copies/mL, regarded as an accepted sensitivity threshold for diagnostic purposes, and reaction specificity was improved as compared to conventional methods. Also notable, these experiments were conducted with conventional reagent concentrations, rather than commonly spiked enzyme and/or template mixtures. This experimental study of the effects of substrate, SA:V, and passivation, together with sensitive and specific microfluidic PCR with consensus degenerate primers represent advances towards lower cost and higher throughput pathogen screening.

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Scanning electron microscopy study of protein immobilized on SIO2 Sol-gel surfaces

Cited by 11 publications

References 6 publications

Characteristics and impact of Taq enzyme adsorption on surfaces in microfluidic devices

Characteristics and impact of Taq enzyme adsorption on surfaces in microfluidic devices

Methods and Supports for Immobilization and Stabilization of Cyclomaltodextrin Glucanotransferase from Thermoanaerobacter

Sensitive, microliter PCR with consensus degenerate primers for Epstein Barr virus amplification

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