Stabilization of α-Chymotrypsin and Lysozyme Entrapped in Water-in-Silicone Oil Emulsions

Zelisko, Paul M.; Brook, Michael A.

doi:10.1021/la025867k

Cited by 41 publications

(55 citation statements)

References 62 publications

(80 reference statements)

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“…That is why amphiphilic polymers are often used as emulsifying agents. A silicone oil necessitates a silicophilic/hydrophilic polymer, such as Abil EM-90 (Cetyl PEG/PPG-10/1 Dimethicone), 29,30 whereas a fluorinated continuous phase requires a fluorophilic/hydrophilic molecule, such as a PFPE-PEG block copolymer (perfluoropolyetherpolyethylene glycol). 31 Regarding biocompatibility, a non-hydrosoluble emulsifying agent is also most suitable to prevent contact of the cells with surfactants.…”

Section: Emulsion Formulationmentioning

confidence: 99%

Micro-confinement of bacteria into w/o emulsion droplets for rapid detection and enumeration

Marcoux

Dupoy

Mathey

et al. 2011

Colloids and Surfaces A: Physicochemical and Engineering Aspect

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Single-cell characterisation and rapid enumeration of E. coli was achieved by confining them into the picoliter droplets of a water-in-oil fuorinated emulsion. Micro-confinement of Bacteria into w/o Emulsion Droplets for Rapid Detection and Enumeration AbstractToday, rapid detection and identification of bacteria in microbiological diagnosis is a major issue. Reference methods usually rely on growth of micro-organisms, with the drawback of lengthy time-to-result. The method provides global information on a clonal population that is known to be inhomogeneous relative to metabolic states and activities. Therefore, there may be a significant advantage of methods that allow characterization of individual bacteria from a large population, both for test time reduction and the clinical value of the characterization. We report here a method for rapid detection and real-time monitoring of the metabolic activities of single bacteria. Water-in-oil emulsions were used to encapsulate single Escherichia coli cells into picolitre (pL)-sized microreactor droplets. The glucuronidase activity in each droplet was monitored using the fluorogenic reporter molecule MUG (4-Methylumbelliferyl β-D-glucuronide) coupled to time-lapse fluorescence imaging of the droplets. Such bacterial confinement provides several major advantages. 2 1) Enzymatic activities of a large number of single bacterium-containing droplet could be monitored simultaneously, allowing the full characterization of metabolic heterogeneity in a clonal population. We monitored glucuronidase enzymatic activity and growth over ~200 single bacteria over a 24h-period. 2) Micro-confinement of cells in small volumes allows rapid accumulation of the fluorescent metabolite, hence decreasing the detection time. Independent of the initial concentration of bacteria in the sample, detection of the presence of bacteria could be achieved in less than two hours. 3) Considering the random distribution of bacteria in droplets, this method allowed rapid and reliable enumeration of bacteria in the initial sample. Overall, the results of this study showed that confinement of bacterial cells increased the effective concentration of fluorescent metabolites leading to rapid (2 h) detection of the fluorescent metabolites, thus significantly reducing time to numeration.

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Section: Emulsion Formulationmentioning

confidence: 99%

Micro-confinement of bacteria into w/o emulsion droplets for rapid detection and enumeration

Marcoux

Dupoy

Mathey

et al. 2011

Colloids and Surfaces A: Physicochemical and Engineering Aspect

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“…The general observation is that the proteins are protected by silicones possessing an optimum balance of a hydrophilic/lipophilic environment [16]. Furthermore, in aspartic proteases, 17 invariant water molecules were found to maintain the structural stability of the enzyme in its native conformation [27].…”

Section: The Activity Of Free and Immobilized Pepsin In Organic Solventsmentioning

confidence: 99%

“…Furthermore the bound fibrinogen was found to be coagulable, as indicated by the thrombin dependent agglutination of the protein coated PDMS droplets [15]. For water-in-oil emulsions, Zelisko and Brook [16] have demonstrated that the presence of silicones bearing hydrophilic groups (silicone surfactants) leads to α-chymotrypsin and lysozyme being stable against denaturation.…”

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confidence: 99%

Immobilization and Activity of Pepsin in Silicone Elastomers

Poojari

Palsule

Clarson

et al. 2009

Silicon

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Pepsin [EC, 3.4.23.1] from Porcine stomach mucosa was immobilized in silicone elastomers utilizing condensation-cure room temperature vulcanization (RTV) of silanol-terminated poly(dimethylsiloxane) (PDMS). Two network precursor chain molar masses were used in this investigation: in pepsin-silicone (A), M n ∼26,000 g mol −1 and in pepsin-silicone (B) M n ∼750 g mol −1 . Tetraethyl orthosilicate (TEOS) was used as the cross-linking agent and dibutyltin dilaurate was used as the catalyst. The activity and stability of free pepsin and pepsin immobilized in PDMS were studied with respect to pH, temperature, cross-link density, solvents and storage time using a hemoglobin assay. A notable finding is that free pepsin has zero activity in neutral buffer solution (pH 7) after incubation for 5 h, while pepsin immobilized in the silicone elastomers was found to retain more than 70% of its maximum normalized activity. There was no marked improvement in the thermal stability of the PDMS immobilized pepsin when compared to free pepsin and all the three systems showed no activity at and above 70°C. From the Lineweaver-Burk kinetic analyses, the apparent K m (g L −1 hemoglobin) for free pepsin was 4.5, for pepsinsilicone (A) was 5.1, and for pepsin-silicone (B) was 3.9, the V max (U/mg of pepsin) for free pepsin was 14,000, for pepsin-silicone (A) was 11,710, and for pepsin-silicone (B) was 8,510, respectively after incubation in buffer solution at pH 2 and 37°C. The activity of the free and the PDMS immobilized pepsin in six different organic solvents was also studied. The pepsin retained high activity in non-polar solvents such as n-hexane, isooctane and toluene, but the enzyme performed poorly in methanol, ethanol and tetrahydrofuran. The degree of swelling of the pepsin immobilized silicone elastomers in these solvents had no impact on the activity of the pepsin. When stored at room temperature for time periods up to 6 months, pepsin immobilized in silicone elastomers was observed to retain its full activity. The results reported herein demonstrate that cross-linked PDMS is a promising support material for the immobilization of hydrolytic enzymes such as pepsin.

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“…To better understand the role that silicone structure, particularly molecular weight, may have on protein denaturation we have undertaken a study of human serum albumin (HSA) denaturation and aggregation as a function of agitation intensity, silicone type (cyclic vs. linear, hydrophobic vs. surface active), protein concentration and, in particular, the molecular weights and molecular weight distributions of the silicone oils. HSA is a particularly convenient protein to study, as we and others have shown, because of the changes in fluorescence maxima that accompany denaturation [13,[18][19][20][21].…”

Section: Introductionmentioning

confidence: 96%

Low molecular weight silicones particularly facilitate human serum albumin denaturation

Nayef

Khan

Brook

2015

Colloids and Surfaces B: Biointerfaces

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Stabilization of α-Chymotrypsin and Lysozyme Entrapped in Water-in-Silicone Oil Emulsions

Cited by 41 publications

References 62 publications

Micro-confinement of bacteria into w/o emulsion droplets for rapid detection and enumeration

Micro-confinement of bacteria into w/o emulsion droplets for rapid detection and enumeration

Immobilization and Activity of Pepsin in Silicone Elastomers

Low molecular weight silicones particularly facilitate human serum albumin denaturation

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