Dynamic passivation with BSA overcomes LTCC mediated inhibition of PCR

Besecker, Jason; Cornell, Ken; Hampikian, Greg

doi:10.1016/j.snb.2012.09.110

Cited by 10 publications

(5 citation statements)

References 29 publications

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“…BSA has advantageous properties such as natural abundance, simple coating procedure, low cost, well-established purification methods, and broad availability. Indeed, BSA coatings-often in the form of a single protein monolayer-have long been used in numerous biological assays, including enzyme-linked immunosorbent assays (ELISA) 7 , blots 8 , immunohistochemistry 9 , and polymerase chain reaction 10 , in order to minimize nonspecific binding events and thus enhance assay performance. In recent years, BSA has also found extensive usage as an antifouling coating material in biosensing 11 and nanomedicine 12 applications.…”

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confidence: 99%

Conformational flexibility of fatty acid-free bovine serum albumin proteins enables superior antifouling coatings

et al. 2020

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Bovine serum albumin (BSA) protein is widely used to fabricate antifouling coatings for nanobiotechnology applications. Numerous BSA protein options are commercially available and obtained through different purification methods, however, there is no guidance on which ones are preferable for antifouling coatings. Herein, we investigate the real-time fabrication of antifouling coatings composed of BSA proteins obtained through different purification methods, and report significant differences in ultrathin film coating properties and ability to mitigate serum biofouling and to prevent nanoparticle-induced immune reactions. Mechanistic studies unravel the source of these performance variations, which are related to some BSA proteins containing fatty acid stabilizers while other BSA proteins are fatty acid-free depending on the purification method. Fatty acid-free BSA proteins exhibit greater conformational flexibility and less charge repulsion, which allow them to form more rigidly attached and tightly packed coatings on flat surfaces and nanoparticles that result in superior application performance.

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confidence: 99%

Conformational flexibility of fatty acid-free bovine serum albumin proteins enables superior antifouling coatings

et al. 2020

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“…9 Here, instead of using the commonly practiced pre-blocking methods for antibody microarray, we used a dynamic passivation method by adding BSA directly to the antigen solution. 27,28 This method circumvents the need for complicated buffer exchange steps. Based on our results, the addition of 1 mg/ml of BSA into the antigen solution would be sufficient for detection level of 0.1 nM of fluorescent proteins in 10 min.…”

Section: Methodsmentioning

confidence: 99%

Multiplexed immunosensing and kinetics monitoring in nanofluidic devices with highly enhanced target capture efficiency

et al. 2016

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Nanofluidic devices promise high reaction efficiency and fast kinetic responses due to the spatial constriction of transported biomolecules with confined molecular diffusion. However, parallel detection of multiple biomolecules, particularly proteins, in highly confined space remains challenging. This study integrates extended nanofluidics with embedded protein microarray to achieve multiplexed real-time biosensing and kinetics monitoring. Implementation of embedded standard-sized antibody microarray is attained by epoxy-silane surface modification and a room-temperature low-aspect-ratio bonding technique. An effective sample transport is achieved by electrokinetic pumping via electroosmotic flow. Through the nanoslit-based spatial confinement, the antigen-antibody binding reaction is enhanced with $100% efficiency and may be directly observed with fluorescence microscopy without the requirement of intermediate washing steps. The image-based data provide numerous spatially distributed reaction kinetic curves and are collectively modeled using a simple one-dimensional convection-reaction model. This study represents an integrated nanofluidic solution for real-time multiplexed immunosensing and kinetics monitoring, starting from device fabrication, protein immobilization, device bonding, sample transport, to data analysis at P eclet number less than 1. Published by AIP Publishing.

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“…The inlet and outlet holes were punched onto the first layer with a biopsy punch (1 mm). The layers were aligned and bonded to each other through oxygen plasma treatment (CUTE-MPR, Femto science, Hwasung, Korea) at 60 watts for 30 s. Finally, the channel and chamber surface were passivated with BSA by filling 100 µL of BSA (50 µg/mL) in PBS (pH 7.4) into the µFPNAS and incubating at 80 • C for 1 h to inhibit non-specific absorption of Ab-MNPs and MSBs into the PDMS surface [36].…”

Section: Microfabrication and Assembly Of Microfluidic Preconcentratimentioning

confidence: 99%

Integrated Microfluidic Preconcentration and Nucleic Amplification System for Detection of Influenza A Virus H1N1 in Saliva

et al. 2020

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Influenza A viruses are often present in environmental and clinical samples at concentrations below the limit of detection (LOD) of molecular diagnostics. Here we report an integrated microfluidic preconcentration and nucleic amplification system (µFPNAS) which enables both preconcentration of influenza A virus H1N1 (H1N1) and amplification of its viral RNA, thereby lowering LOD for H1N1. H1N1 virus particles were first magnetically preconcentrated using magnetic nanoparticles conjugated with an antibody specific for the virus. Their isolated RNA was amplified to cDNA through thermocycling in a trapezoidal chamber of the µFPNAS. A detection limit as low as 100 TCID50 (50% tissue culture infective dose) in saliva can be obtained within 2 hours. These results suggest that the LOD of molecular diagnostics for virus can be lowered by systematically combining immunomagnetic separation and reverse transcriptase-polymerase chain reaction (RT-PCR) in one microfluidic device.

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Dynamic passivation with BSA overcomes LTCC mediated inhibition of PCR

Cited by 10 publications

References 29 publications

Conformational flexibility of fatty acid-free bovine serum albumin proteins enables superior antifouling coatings

Conformational flexibility of fatty acid-free bovine serum albumin proteins enables superior antifouling coatings

Multiplexed immunosensing and kinetics monitoring in nanofluidic devices with highly enhanced target capture efficiency

Integrated Microfluidic Preconcentration and Nucleic Amplification System for Detection of Influenza A Virus H1N1 in Saliva

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