Novel RP-HPLC based assay for selective and sensitive endotoxin quantification

Bucsella, Blanka; Hoffmann, Anika; Zollinger, Mathieu; Stephan, F; Pattky, Martin; Daumke, Ralph; Heiligtag, Florian J; Frank, Brian P.; Bassas-Galià, Mònica; Zinn, Manfred; Kálmán, Franka

doi:10.1039/d0ay00872a

Cited by 9 publications

(6 citation statements)

References 49 publications

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“…To determine the amount of glycan conjugated to gold nanoparticles, we used an exo‐glycosidase to release the non‐reducing terminal sugar of the glycan on the particle followed by quantitative analysis of the released sugar. Since G10 has a terminal sialic acid, we used neuraminidase to release the terminal sialic acid which was then reacted with 1,2‐diamino‐4,5‐methylenedioxybenzene (DMB) to form a fluorescent adduct [31] ( Scheme 2) for the quantitative measure of the amount of glycan conjugated to the particles.…”

Section: Resultsmentioning

confidence: 99%

Synthesis and Quantitative Analysis of Glycans Conjugated to Gold Nanoparticles

Liao,

Kung,

Cheng

et al. 2024

Helvetica Chimica Acta

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Nanoparticles, especially gold nanoparticles (GNPs) have emerged as promising tools for biomedical applications due to their unique properties and ability to be functionalized. However, quantitative analysis of biomolecules conjugated to nanoparticles remains a challenge. Here we report a method to conjugate a synthetic hybrid‐type N‐glycan to GNPs and quantitatively analyze the glycan content. The glycan was first conjugated to N‐hydroxysuccinimide (NHS)‐ester activated GNPs and confirmed qualitatively by Fourier‐transform infrared spectroscopy and flow cytometry. The glycan conjugated‐GNPs were then treated with neuraminidase to release terminal sialic acid from the glycan, which was labeled with 1,2‐diamino‐4,5‐methylenedioxybenzene and quantitatively analyzed by Ultraperformance Liquid Chromatography (UPLC). The amount of the sialic acid released was equivalent to the glycan content on GNPs. This method enabled a precise quantification of glycans conjugated to gold nanoparticles and should facilitate its biomedical applications, including studies of multivalent receptor‐glycan interaction, cell targeting and sorting, and immunization. Overall, this work provides an effective approach to synthesize and characterize nanoparticles conjugates.

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Section: Resultsmentioning

confidence: 99%

Synthesis and Quantitative Analysis of Glycans Conjugated to Gold Nanoparticles

Liao,

Kung,

Cheng

et al. 2024

Helvetica Chimica Acta

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“…The development of the Kdo-DMB-LC ET assay is described in detail in Bucsella et al [28] for HPLC (Agilent 1260 infinity II, Agilent Technologies, USA) and was performed as described there. In short: Two µL of acetic acid were added to 100 µL sample aliquots.…”

Section: Kdo-dmb Liquid Chromatography Assaymentioning

confidence: 99%

“…Aliquots of 100 µL were stored at − 20 • C until use. The external ET standards were subjected to hydrolysis and derivatization with DMB in parallel to bioreactor samples as described in Bucsella et al [28]. The ET concentration of a bioreactor sample was calculated by comparing the signal intensity obtained for the 500 ng mL − 1 ET standard to signal intensities obtained for bioreactor samples.…”

Section: Calibration With External Endotoxin Standardsmentioning

confidence: 99%

“…Recently, our group reported a novel chemical instrumental analytical ET quantification assay [28]. It is based on the ET-specific marker Kdo [28][29][30].…”

Section: Introductionmentioning

confidence: 99%

“…Recently, our group reported a novel chemical instrumental analytical ET quantification assay [28]. It is based on the ET-specific marker Kdo [28][29][30]. The sugar acid Kdo connects the highly conserved core region of an ET to Lipid A.…”

Section: Introductionmentioning

confidence: 99%

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