Development of a Sensitive Quantum Dot-Linked Immunoassay for the Multiplex Detection of Biochemical Markers in a Microvolumeric Format
Kalvaityte, Ursule; Bagdonas, Edvardas; Kirdaite, Gailute; Kausaite-Minkstimiene, Asta; Uzieliene, Ilona; Ramanaviciene, Almira; Popov, Anton; Butkiene, Greta; Karabanovas, Vitalijus; Denkovskij, Jaroslav; Mobasheri, Ali; Bernotiene, Eiva (2025-02-07)
Dove Medical Press
07.02.2025
Kalvaityte, U., Bagdonas, E., Kirdaite, G., Kausaite-Minkstimiene, A., Uzieliene, I., Ramanaviciene, A., Popov, A., Butkiene, G., Karabanovas, V., Denkovskij, J., Mobasheri, A. & Bernotiene, E. (2025) Development of a Sensitive Quantum Dot-Linked Immunoassay for the Multiplex Detection of Biochemical Markers in a Microvolumeric Format, International journal of nanomedicine, 20, 1717-1729, DOI: 10.2147/IJN.S477118
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© 2025 Kalvaityte et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php.
https://creativecommons.org/licenses/by-nc/3.0/
© 2025 Kalvaityte et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php.
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Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:oulu-202502131619
https://urn.fi/URN:NBN:fi:oulu-202502131619
Tiivistelmä
Abstract
Purpose:
For the diagnosis of various diseases, simultaneous sensitive detection of multiple biomarkers using low sample volumes is needed. The purpose of the present research was to develop sensitive multiplex detection model of QD-based ELISA (QLISA), through the spectroscopic QD-analyte complex measurements in microvolume liquid droplets on a glass microslide.
Methods:
QLISA was used for the detection of cartilage oligomeric matrix protein (COMP) and human growth hormone (hGH) as model analytes. The QLISA detection method included the formation of complexes consisting of analyte antigens, biotinylated antibodies and streptavidin-coated QDs. A specific immune-complex disassembling solution was used to dissociate analyte-antibody complexes from the bottom of the 96-well plate. After dissociation, the samples were diluted with PBS, and 2 μL transferred to a reusable glass slide for fluorescence (FL) scan.
Results:
The alkaline immune-complex disassembling solution that most efficiently amplified QDs FL within a prolonged 17 h time was selected. Comparison of median fluorescence intensity (MFI) of 50 nM COMP, 25 nM COMP, and 5 nM COMP detection using QD655 with the dilution of the detached samples with PBS and without dilution resulted in significant MFI differences in all cases. The FL signal readouts from QD655 in the microvolume format were from 10 to 40 times stronger than those measured directly from a 96-well plate QLISAs. In duplex analysis, two analytes COMP and hGH were measured using different QD605 and QD525 in the same well. In the respectful 96-well plate QLISA format, two different analyte concentrations can be hardly distinguishable, but the transfer to micro-volumetric detection on the glass slide highly increased the signal strength according to green and red FL intensity of QDs.
Conclusion:
Our method significantly enhances detection sensitivity, as compared to measured in parallel QLISAs in a 96 well plate format, enables multiplexing and may prove very valuable for samples of limited volumes.
Purpose:
For the diagnosis of various diseases, simultaneous sensitive detection of multiple biomarkers using low sample volumes is needed. The purpose of the present research was to develop sensitive multiplex detection model of QD-based ELISA (QLISA), through the spectroscopic QD-analyte complex measurements in microvolume liquid droplets on a glass microslide.
Methods:
QLISA was used for the detection of cartilage oligomeric matrix protein (COMP) and human growth hormone (hGH) as model analytes. The QLISA detection method included the formation of complexes consisting of analyte antigens, biotinylated antibodies and streptavidin-coated QDs. A specific immune-complex disassembling solution was used to dissociate analyte-antibody complexes from the bottom of the 96-well plate. After dissociation, the samples were diluted with PBS, and 2 μL transferred to a reusable glass slide for fluorescence (FL) scan.
Results:
The alkaline immune-complex disassembling solution that most efficiently amplified QDs FL within a prolonged 17 h time was selected. Comparison of median fluorescence intensity (MFI) of 50 nM COMP, 25 nM COMP, and 5 nM COMP detection using QD655 with the dilution of the detached samples with PBS and without dilution resulted in significant MFI differences in all cases. The FL signal readouts from QD655 in the microvolume format were from 10 to 40 times stronger than those measured directly from a 96-well plate QLISAs. In duplex analysis, two analytes COMP and hGH were measured using different QD605 and QD525 in the same well. In the respectful 96-well plate QLISA format, two different analyte concentrations can be hardly distinguishable, but the transfer to micro-volumetric detection on the glass slide highly increased the signal strength according to green and red FL intensity of QDs.
Conclusion:
Our method significantly enhances detection sensitivity, as compared to measured in parallel QLISAs in a 96 well plate format, enables multiplexing and may prove very valuable for samples of limited volumes.
Kokoelmat
- Avoin saatavuus [38865]
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