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Water-soluble electrospun nanofibers as a method for on-chip reagent storage.

Dai M, Jin S, Nugen SR - Biosensors (Basel) (2012)

Bottom Line: This work demonstrates the ability to electrospin reagents into water-soluble nanofibers resulting in a stable on-chip enzyme storage format.Immediately following electrospinning, the activity peak for the HRP decreased by approximately 20%.The ability to store enzymes and other reagents on-chip in a rapidly dispersible format could reduce the assay steps required of an operator to perform.

View Article: PubMed Central - PubMed

Affiliation: University of Massachusetts, 102 Holdsworth Way, Amherst, MA 01003, USA. mdai@foodsci.umass.edu.

ABSTRACT
This work demonstrates the ability to electrospin reagents into water-soluble nanofibers resulting in a stable on-chip enzyme storage format. Polyvinylpyrrolidone (PVP) nanofibers were spun with incorporation of the enzyme horseradish peroxidase (HRP). Scanning electron microscopy (SEM) of the spun nanofibers was used to confirm the non-woven structure which had an average diameter of 155 ± 34 nm. The HRP containing fibers were tested for their change in activity following electrospinning and during storage. A colorimetric assay was used to characterize the activity of HRP by reaction with the nanofiber mats in a microtiter plate and monitoring the change in absorption over time. Immediately following electrospinning, the activity peak for the HRP decreased by approximately 20%. After a storage study over 280 days, 40% of the activity remained. In addition to activity, the fibers were observed to solubilize in the microfluidic chamber. The chromogenic 3,3',5,5'-tetramethylbenzidine solution reacted immediately with the fibers as they passed through a microfluidic channel. The ability to store enzymes and other reagents on-chip in a rapidly dispersible format could reduce the assay steps required of an operator to perform.

No MeSH data available.


(A) Horseradish peroxidase (HRP) activity before and after electrospinning was detected by a 1-step slow TMB kit. 150μL 1-step slow TMB kit mixed with 100μL water was used by measuring the change in absorption at 652nm every 15 s for 1 h. The equivalent quantity of HRP was 0.04 μg. The reaction initially oxidized the TMB substrate yielding a blue color at 652 nm peak in absorbance. As the reaction progressed, the color shifted to yellow and had maximal absorbance at 450 nm. (B) The rate of inactivation was rapid at first and slowed over time. After 280 days, the enzyme activity was approximately 40%.
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biosensors-02-00388-f002: (A) Horseradish peroxidase (HRP) activity before and after electrospinning was detected by a 1-step slow TMB kit. 150μL 1-step slow TMB kit mixed with 100μL water was used by measuring the change in absorption at 652nm every 15 s for 1 h. The equivalent quantity of HRP was 0.04 μg. The reaction initially oxidized the TMB substrate yielding a blue color at 652 nm peak in absorbance. As the reaction progressed, the color shifted to yellow and had maximal absorbance at 450 nm. (B) The rate of inactivation was rapid at first and slowed over time. After 280 days, the enzyme activity was approximately 40%.

Mentions: The average weight of each 1 cm fiber mat was 0.8 mg and was subsequently calculated to contain approximately 0.04 μg HRP. Therefore, during activity measurements, the control contained 0.04 μg HRP with similar ratios of PVP and sucrose. Following the addition of TMB-containing reaction solution to the nanofibers, the absorbance at 652 nm was monitored over time Figure 2(A). The results indicate that the HRP control solution without having been electrospun peaked at 30 s and then sharply declined. The average maximum absorption for the control was 1.21. The absorption peak of the electrospun nanofibers was 0.99 which occurred only after 1,300 s. This is most likely due to slower hydration in the presence of the PVP matrix. Part of the slow hydration may have been caused by bunching of the mat which had a relatively large area being placed into a relatively small well. Following desiccated storage at room temperature for 45 days, the activity of the electrospun HRP decreased to 60% and decreased further to 40% after 280 days. The rate at which the enzyme lost activity appeared to decrease over time suggesting possible stabilization (Figure 2(B)).


Water-soluble electrospun nanofibers as a method for on-chip reagent storage.

Dai M, Jin S, Nugen SR - Biosensors (Basel) (2012)

(A) Horseradish peroxidase (HRP) activity before and after electrospinning was detected by a 1-step slow TMB kit. 150μL 1-step slow TMB kit mixed with 100μL water was used by measuring the change in absorption at 652nm every 15 s for 1 h. The equivalent quantity of HRP was 0.04 μg. The reaction initially oxidized the TMB substrate yielding a blue color at 652 nm peak in absorbance. As the reaction progressed, the color shifted to yellow and had maximal absorbance at 450 nm. (B) The rate of inactivation was rapid at first and slowed over time. After 280 days, the enzyme activity was approximately 40%.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263557&req=5

biosensors-02-00388-f002: (A) Horseradish peroxidase (HRP) activity before and after electrospinning was detected by a 1-step slow TMB kit. 150μL 1-step slow TMB kit mixed with 100μL water was used by measuring the change in absorption at 652nm every 15 s for 1 h. The equivalent quantity of HRP was 0.04 μg. The reaction initially oxidized the TMB substrate yielding a blue color at 652 nm peak in absorbance. As the reaction progressed, the color shifted to yellow and had maximal absorbance at 450 nm. (B) The rate of inactivation was rapid at first and slowed over time. After 280 days, the enzyme activity was approximately 40%.
Mentions: The average weight of each 1 cm fiber mat was 0.8 mg and was subsequently calculated to contain approximately 0.04 μg HRP. Therefore, during activity measurements, the control contained 0.04 μg HRP with similar ratios of PVP and sucrose. Following the addition of TMB-containing reaction solution to the nanofibers, the absorbance at 652 nm was monitored over time Figure 2(A). The results indicate that the HRP control solution without having been electrospun peaked at 30 s and then sharply declined. The average maximum absorption for the control was 1.21. The absorption peak of the electrospun nanofibers was 0.99 which occurred only after 1,300 s. This is most likely due to slower hydration in the presence of the PVP matrix. Part of the slow hydration may have been caused by bunching of the mat which had a relatively large area being placed into a relatively small well. Following desiccated storage at room temperature for 45 days, the activity of the electrospun HRP decreased to 60% and decreased further to 40% after 280 days. The rate at which the enzyme lost activity appeared to decrease over time suggesting possible stabilization (Figure 2(B)).

Bottom Line: This work demonstrates the ability to electrospin reagents into water-soluble nanofibers resulting in a stable on-chip enzyme storage format.Immediately following electrospinning, the activity peak for the HRP decreased by approximately 20%.The ability to store enzymes and other reagents on-chip in a rapidly dispersible format could reduce the assay steps required of an operator to perform.

View Article: PubMed Central - PubMed

Affiliation: University of Massachusetts, 102 Holdsworth Way, Amherst, MA 01003, USA. mdai@foodsci.umass.edu.

ABSTRACT
This work demonstrates the ability to electrospin reagents into water-soluble nanofibers resulting in a stable on-chip enzyme storage format. Polyvinylpyrrolidone (PVP) nanofibers were spun with incorporation of the enzyme horseradish peroxidase (HRP). Scanning electron microscopy (SEM) of the spun nanofibers was used to confirm the non-woven structure which had an average diameter of 155 ± 34 nm. The HRP containing fibers were tested for their change in activity following electrospinning and during storage. A colorimetric assay was used to characterize the activity of HRP by reaction with the nanofiber mats in a microtiter plate and monitoring the change in absorption over time. Immediately following electrospinning, the activity peak for the HRP decreased by approximately 20%. After a storage study over 280 days, 40% of the activity remained. In addition to activity, the fibers were observed to solubilize in the microfluidic chamber. The chromogenic 3,3',5,5'-tetramethylbenzidine solution reacted immediately with the fibers as they passed through a microfluidic channel. The ability to store enzymes and other reagents on-chip in a rapidly dispersible format could reduce the assay steps required of an operator to perform.

No MeSH data available.