The pros and cons of nucleic acid-amplified immunoassays—a comparative study on the quantitation of prostate-specific antigen with and without rolling circle amplification

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216224%3A14310%2F24%3A00139615" target="_blank" >RIV/00216224:14310/24:00139615 - isvavai.cz</a>

Výsledek na webu

<a href="https://link.springer.com/article/10.1007/s00216-024-05357-y" target="_blank" >https://link.springer.com/article/10.1007/s00216-024-05357-y</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1007/s00216-024-05357-y" target="_blank" >10.1007/s00216-024-05357-y</a>

Jazyk výsledku

angličtina

Název v původním jazyce

The pros and cons of nucleic acid-amplified immunoassays—a comparative study on the quantitation of prostate-specific antigen with and without rolling circle amplification

Popis výsledku v původním jazyce

Integrating isothermal nucleic acid amplification strategies into immunoassays can significantly decrease analytical limits of detection (LODs). On the other hand, an amplification step adds time, complication, reagents, and costs to the assay format. To evaluate the pros and cons in the context of heterogeneous multistep immunoassays, we quantified prostate-specific antigen (PSA) with and without rolling circle amplification (RCA). In addition, we compared time-gated (TG) with continuous-wave (CW) photoluminescence (PL) detection using a terbium complex and a fluorescein dye, respectively. For both direct (non-amplified) and amplified assays, TG PL detection provided circa four- to eightfold lower LODs, illustrating the importance of autofluorescence background suppression even for multi-wash assay formats. Amplified assays required an approximately 2.4 h longer assay time but led to almost 100-fold lower LODs down to 1.3 pg/mL of PSA. Implementation of TG-FRET (using a Tb-Cy5.5 donor–acceptor pair) into the RCA immunoassay resulted in a slightly higher LOD (3.0 pg/mL), but the ratiometric detection format provided important benefits, such as higher reproducibility, lower standard deviations, and multiplexing capability. Overall, our direct comparison demonstrated the importance of biological background suppression even in heterogeneous assays and the potential of using isothermal RCA for strongly decreasing analytical LODs, making such assays viable alternatives to conventional enzyme-linked immunosorbent assays (ELISAs).

Název v anglickém jazyce

The pros and cons of nucleic acid-amplified immunoassays—a comparative study on the quantitation of prostate-specific antigen with and without rolling circle amplification

Popis výsledku anglicky

Integrating isothermal nucleic acid amplification strategies into immunoassays can significantly decrease analytical limits of detection (LODs). On the other hand, an amplification step adds time, complication, reagents, and costs to the assay format. To evaluate the pros and cons in the context of heterogeneous multistep immunoassays, we quantified prostate-specific antigen (PSA) with and without rolling circle amplification (RCA). In addition, we compared time-gated (TG) with continuous-wave (CW) photoluminescence (PL) detection using a terbium complex and a fluorescein dye, respectively. For both direct (non-amplified) and amplified assays, TG PL detection provided circa four- to eightfold lower LODs, illustrating the importance of autofluorescence background suppression even for multi-wash assay formats. Amplified assays required an approximately 2.4 h longer assay time but led to almost 100-fold lower LODs down to 1.3 pg/mL of PSA. Implementation of TG-FRET (using a Tb-Cy5.5 donor–acceptor pair) into the RCA immunoassay resulted in a slightly higher LOD (3.0 pg/mL), but the ratiometric detection format provided important benefits, such as higher reproducibility, lower standard deviations, and multiplexing capability. Overall, our direct comparison demonstrated the importance of biological background suppression even in heterogeneous assays and the potential of using isothermal RCA for strongly decreasing analytical LODs, making such assays viable alternatives to conventional enzyme-linked immunosorbent assays (ELISAs).

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10406 - Analytical chemistry

Projekt

<a href="/cs/project/GA22-27580S" target="_blank" >GA22-27580S: Laserová spektroskopie v imunostanovení a zobrazování s nanometalickými značkami</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2024

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Analytical and Bioanalytical Chemistry

ISSN

1618-2642

e-ISSN

1618-2650

Svazek periodika

416

Číslo periodika v rámci svazku

30

Stát vydavatele periodika

DE - Spolková republika Německo

Počet stran výsledku

10

Strana od-do

7285-7294

Kód UT WoS článku

001242267600001

EID výsledku v databázi Scopus

2-s2.0-85195402714