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bioRxiv · 10.1101/2021.02.25.432859

Development of a label-free microfluidic impedimetric immunoassay for anti-SPAG16 antibody.

Froyen, T.·Vreys, F.·Thoelen, R.·Slaets, L.·Somers, V.
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Abstract

A recently found biomarker regarding multiple sclerosis, namely the anti-SPAG16 antibody (Ab), could be a potential way for early detection, prognosis and diagnosis of said autoimmune disease. Merging electrochemical analysis with a microfluidic system is a novel approach, which avoids the use of labelling steps as seen in traditional ELISA immunoassays. In this study, aluminium interdigitated electrodes on polystyrene-coated PET foils were implemented in a microfluidic flow cell to bind and detect SPAG16 Abs by impedimetric measurements. The coated PET foils showed a clear affinity for the fusion protein SPAG16-THIO and thioredoxin (THIO). Determining sensitivity and specificity of antibody-antigen binding using a microfluidic ELISA immunoassay has revealed the test to be unreliable by showing no linear pattern of a dilution series of the standard and producing skewed inconsistent results. The impedimetric analysis showed opposite results of what one would expect. The systems efficiency is in need to be revised and optimised before undergoing actual diagnostic tests. Further advancement could be done by reducing leakage, securing a more stable entrance for injection and circumventing the occurence of air bubbles in the wells. SAMENVATTINGMultiple sclerosis (MS) is een auto-immune ziekte, gekenmerkt door inflammatie van het centraal zenuwstelsel en demyelinisatie van axonen. Een recent gevonden proteine, namelijk het anti-SPAG16 antilichaam, is bewezen een biomerker te zijn voor MS. Met behulp van een elektrochemisch analytisch systeem gecombineerd met microfluidica zou men anti-SPAG16 antilichamen kunnen opsporen voor vroegtijdige detectie, prognose en diagnose. Het grote voordeel tegenover traditionele ELISA testen is het elimineren van labelling stappen waardoor de immunoassay goedkoper en gebruiksvriendelijk wordt. In deze studie werden overlappende aluminium elektrodes op polystyreen-gecoate PET-plaatjes geimplementeerd in een microfluidische flow cell voor binding en detectie van anti-SPAG16 antilichamen door middel van impedimetrische metingen. De gecoate PET-plaatjes vertonen een duidelijke affiniteit voor 0,1 g/ml SPAG16 en THIO. Het bepalen van sensitiviteit en specificiteit van antilichaam-antigen binding, gebruikmakende van een ELISA immunoassays, gaf aan dat de test onbetrouwbaar is doordat geen duidelijk patroon voor standaardoplossingen zichtbaar was en resultaten vertekend en inconsistent bleken te zijn. De impedimetrische analyse vertoonde tevens onbetrouwbare resultaten, waarbij een omgekeerd effect werd geobserveerd van wat er in theorie zou moeten gebeuren. De efficientie van het systeem moet herzien en geoptimaliseerd worden voordat men het voor diagnostische testen kan gebruiken. Verdere vorderingen zouden gerealiseerd kunnen worden door lekkages te reduceren, steviger bevestigen van de PDMS-ingang en de ontwikkeling van luchtbellen in de wells te vermijden.

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Froyen, T., Vreys, F., Thoelen, R., Slaets, L., Somers, V.. 2021-02-26. Development of a label-free microfluidic impedimetric immunoassay for anti-SPAG16 antibody.. https://doi.org/10.1101/2021.02.25.432859

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