A novel low-cost approach for the semi-quantitative analysis of carbohydrate-deficient transferrin (CDT) based on fluorescence resonance energy transfer (FRET)

Musile, Giacomo; Palo, Elio Franco De; Savchuk, S. A.; Shestakova, Ksenia; Bortolotti, Federica; Tagliaro, Franco

doi:10.1016/j.cca.2019.06.003

Cited by 6 publications

(4 citation statements)

References 17 publications

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“…Electrostatic interactions between arginine containing FAM and negatively charged CNPs shorten the distance allowing the transfer of fluorescent energy from FAM to CNPs [ 40 , 41 ]. FAM was excited as a fluorophore and became an energy donor, and the fluorescence was quenched by almost 100% after adding CNPs, which indicated that the energy difference between the vibrational levels of the donor FAM’s ground state and the first excited state and the vibrational levels of the acceptor CNPs had a high degree of overlap [ 42 , 43 ].…”

Section: Resultsmentioning

confidence: 99%

A carbon nanoparticle-peptide fluorescent sensor custom-made for simple and sensitive detection of trypsin

Hou

Feng

Zhao

et al. 2020

Journal of Pharmaceutical Analysis

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Herein, we report a novel sensor to detect trypsin using a purpose-designed fluorescein-labelled peptide with negatively charged carbon nanoparticles (CNPs) modified by acid oxidation. The fluorescence of the fluorescein-labelled peptide was quenched by CNPs. The sensor reacted with trypsin to cleave the peptide, resulting in the release of the dye moiety and a substantial increase in fluorescence intensity, which was dose- and time-dependent, and trypsin could be quantified accordingly. Correspondingly, the biosensor has led to the development of a convenient and efficient fluorescent method to measure trypsin activity, with a detection limit of 0.7 μg/mL. The method allows rapid determination of trypsin activity in the normal and acute pancreatitis range, suitable for point-of-care testing. Furthermore, the applicability of the method has been demonstrated by detecting trypsin in spiked urine samples.

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Section: Resultsmentioning

confidence: 99%

A carbon nanoparticle-peptide fluorescent sensor custom-made for simple and sensitive detection of trypsin

Hou

Feng

Zhao

et al. 2020

Journal of Pharmaceutical Analysis

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“…The Tb 3+ fluorescence setup, already optimized in previous works for transferrin (Tf) [ 27 , 28 , 29 ]—the Lf serum homologous—was reproduced here for commercial Lf. Standard solutions of Lf were prepared by solubilization of commercial Lf powders (Apo-Lf and Fe 3+ -Lf) in 20 mM ammonium bicarbonate, pH 8, and 100 mM ammonium acetate.…”

Section: Methodsmentioning

confidence: 99%

Contact Lens Wear Induces Alterations of Lactoferrin Functionality in Human Tears

Ponzini

Tavazzi²,

Musile³

et al. 2022

Pharmaceutics

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The tear film is a complex matrix composed of several molecular classes, from small metal ions to macromolecules. Contact lens (CL) wear can affect the protein homeostasis of the tear film, by accumulating deposits on the CL surface and/or altering their structural and functional properties. This work investigates the effect of CL wear on lactoferrin (Lf), one of the most abundant tear proteins, known as an unspecific biomarker of inflammation. Tears from eight volunteers were collected and analyzed after alternated periods of CL wear and without CL. The experimental approach is to probe Lf into unprocessed human tears by the peculiar fluorescence emission originating from complex formation of Lf with terbium (Tb3+) at the iron-binding sites. The experimental data indicate that CL wear does not significantly affect the total amount of Lf. On the other hand, Lf affinity for Tb3+ is reduced upon CL wear, suggesting relevant changes in Lf structure and possible alterations of protein functionality. Future studies based on this approach will help define CL features (material, lens-care solution, wearing time, etc.) with minimal effects on tear protein activity, in order to obtain more biocompatible and comfortable devices.

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“…Other methods for CDT analysis have been reported, including those based on capillary zone electrophoresis [3] and HPLC [4], the latter of which is currently the IFCC reference method for CDT measurement. However, the detection modes in these methods are typically based on UV/visible absorption or native fluorescence [5], and, therefore, have limited sensi-tivity. Recently, the sensitivity was improved by harnessing the ability of Tf to bind Tb 3+ , which greatly enhances the intrinsic fluorescence of Tf, resulting in a relevant increase in analytical sensitivity by a factor of approximately 3 [6].…”

Section: Methods Summarymentioning

confidence: 99%

A Proof-of-concept Analysis of Carbohydrate-deficient Transferrin by Imaged Capillary Isoelectric Focusing and In-capillary Immunodetection

Haitjema

Heger

et al. 2020

BioTechniques

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Carbohydrate-deficient transferrin (CDT) is a reliable biomarker for chronic alcohol abuse. We developed a method for CDT analysis by capillary isoelectric focusing, followed by immunodetection directly in the capillary, in an automated fashion and on a single platform (Peggy Sue™; ProteinSimple, CA, USA). Transferrin glycoforms in serum samples, including disialo-transferrin, were separated and their apparent isoelectric points and relative percentages were determined. The relative CDT values (percent of total transferrin) matched expected values for both healthy and alcoholic samples. Because the method leveraged the sensitivity of an immunoassay, CDT was measured when serum samples were diluted up to 1200-fold, reducing the volume of serum required. Finally, the process is fully automated, with up to 96 samples analyzed per batch.

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A novel low-cost approach for the semi-quantitative analysis of carbohydrate-deficient transferrin (CDT) based on fluorescence resonance energy transfer (FRET)

Cited by 6 publications

References 17 publications

A carbon nanoparticle-peptide fluorescent sensor custom-made for simple and sensitive detection of trypsin

A carbon nanoparticle-peptide fluorescent sensor custom-made for simple and sensitive detection of trypsin

Contact Lens Wear Induces Alterations of Lactoferrin Functionality in Human Tears

A Proof-of-concept Analysis of Carbohydrate-deficient Transferrin by Imaged Capillary Isoelectric Focusing and In-capillary Immunodetection

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