Nanomechanical detection of cholera toxin using microcantilevers functionalized with ganglioside nanodiscs

Tark, Soo Hyun; Das, Aditi; Sligar, Stephen G.; Dravid, Vinayak P.

doi:10.1088/0957-4484/21/43/435502

Cited by 25 publications

(20 citation statements)

References 57 publications

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“…Moreover, this technology has enabled the stabilization of membrane proteins both in solution [36][37][38][39][40] and on surfaces. 39,[41][42][43][44]…”

Section: N-terminus Role In Association With Lipid Bilayers Activitymentioning

confidence: 99%

Functional studies of N‐terminally modified CYP2J2 epoxygenase in model lipid bilayers

et al. 2013

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CYP2J2 epoxygenase is a membrane bound cytochrome P450 that converts omega-3 and omega-6 fatty acids into physiologically active epoxides. In this work, we present a comprehensive comparison of the effects of N-terminal modifications on the properties of CYP2J2 with respect to the activity of the protein in model lipid bilayers using Nanodiscs. We demonstrate that the complete truncation of the N-terminus changes the association of this protein with the E.coli membrane but does not disrupt incorporation in the lipid bilayers of Nanodiscs. Notably, the introduction of silent mutations at the N-terminus was used to express full length CYP2J2 in E. coli while maintaining wild-type functionality. We further show that lipid bilayers are essential for the productive use of NADPH for ebastine hydroxylation by CYP2J2. Taken together, it was determined that the presence of the N-terminus is not as critical as the presence of a membrane environment for efficient electron transfer from cytochrome P450 reductase to CYP2J2 for ebastine hydroxylation in Nanodiscs. This suggests that adopting the native-like conformation of CYP2J2 and cytochrome P450 reductase in lipid bilayers is essential for effective use of reducing equivalents from NADPH for ebastine hydroxylation.

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“…Moreover, this technology has enabled the stabilization of membrane proteins both in solution [36][37][38][39][40] and on surfaces. 39,[41][42][43][44]…”

Section: N-terminus Role In Association With Lipid Bilayers Activitymentioning

confidence: 99%

Functional studies of N‐terminally modified CYP2J2 epoxygenase in model lipid bilayers

et al. 2013

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“…For larger toxins, it is possible to use non-competitive [20] and sandwich [70] based biosensor methods; lower molecular weight toxins require inhibition/competition-based methods [71].…”

Section: Toxinsmentioning

confidence: 99%

“…Table 1 [2,7,8,[11][12][13] lists the most common classifications. The following references are recent articles that provide details on the principles of the different types of transducer: electrochemical [13][14][15], optical [15][16][17][18], mass/ acoustic [15,19,20] and thermal [21].…”

Section: Introductionmentioning

confidence: 99%

Biosensors for the analysis of microbiological and chemical contaminants in food

2012

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Increases in food production and the ever-present threat of food contamination from microbiological and chemical sources have led the food industry and regulators to pursue rapid, inexpensive methods of analysis to safeguard the health and safety of the consumer. Although sophisticated techniques such as chromatography and spectrometry provide more accurate and conclusive results, screening tests allow a much higher throughput of samples at a lower cost and with less operator training, so larger numbers of samples can be analysed. Biosensors combine a biological recognition element (enzyme, antibody, receptor) with a transducer to produce a measurable signal proportional to the extent of interaction between the recognition element and the analyte. The different uses of the biosensing instrumentation available today are extremely varied, with food analysis as an emerging and growing application. The advantages offered by biosensors over other screening methods such as radioimmunoassay, enzyme-linked immunosorbent assay, fluorescence immunoassay and luminescence immunoassay, with respect to food analysis, include automation, improved reproducibility, speed of analysis and real-time analysis. This article will provide a brief footing in history before reviewing the latest developments in biosensor applications for analysis of food contaminants (January 2007 to December 2010), focusing on the detection of pathogens, toxins, pesticides and veterinary drug residues by biosensors, with emphasis on articles showing data in food matrices. The main areas of development common to these groups of contaminants include multiplexing, the ability to simultaneously analyse a sample for more than one contaminant and portability. Biosensors currently have an important role in food safety; further advances in the technology, reagents and sample handling will surely reinforce this position.

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“…[ 11,50 ] GpAtm incorporation into the nanodiscs was verified using a biotin labeled GpAtm derivative that induces a significant size increase after the addition of streptavidin, which also allows nanodiscs separation during size exclusion chromatography (Figure S9, Supporting Information). Then, the nanodisc solution with a concentration of 1.6 µg mL −1 was drop cast on a gold substrate, which was prefunctionalized with 3,3′‐dithiobis(sulfosuccinimidyl propionate) (DTSSP) [ 51 ] to enhance binding of the nanodiscs to the gold surface (Figure S6, Supporting Information). The nanodiscs adsorbed at the gold surface (Figure S7, Supporting Information) were then embedded into the PDA film during electropolymerization (Figure 4A).…”

Section: Resultsmentioning

confidence: 99%

Ultrathin Polydopamine Films with Phospholipid Nanodiscs Containing a Glycophorin A Domain

D'Alvise

Harvey

Hueske

et al. 2020

Adv Funct Materials

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Cellular membranes have long served as an inspiration for nanomaterial research. The preparation of ultrathin polydopamine (PDA) films with integrated protein pores containing phospholipids and an embedded domain of a membrane protein glycophorin A as simplified cell membrane mimics is reported. Large area, ultrathin PDA films are obtained by electropolymerization on gold surfaces with 10-18 nm thickness and dimensions of up to 2.5 cm 2 . The films are transferred from gold to various other substrates such as nylon mesh, silicon, or substrates containing holes in the micrometer range, and they remain intact even after transfer. The novel transfer technique gives access to freestanding PDA films that remain stable even at the air interfaces with elastic moduli of ≈6-12 GPa, which are higher than any other PDA films reported before. As the PDA film thickness is within the range of cellular membranes, monodisperse protein nanopores, so-called "nanodiscs," are integrated as functional entities. These nanodisc-containing PDA films can serve as semipermeable films, in which the embedded pores control material transport. In the future, these simplified cell membrane mimics may offer structural investigations of the embedded membrane proteins to receive an improved understanding of protein-mediated transport processes in cellular membranes.

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Nanomechanical detection of cholera toxin using microcantilevers functionalized with ganglioside nanodiscs

Cited by 25 publications

References 57 publications

Functional studies of N‐terminally modified CYP2J2 epoxygenase in model lipid bilayers

Functional studies of N‐terminally modified CYP2J2 epoxygenase in model lipid bilayers

Biosensors for the analysis of microbiological and chemical contaminants in food

Ultrathin Polydopamine Films with Phospholipid Nanodiscs Containing a Glycophorin A Domain

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