Search bioRxiv⌕ Search
Home/Archive/bioRxiv

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.
View original source ↗Export metadata ↓

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.

Explore related subjects

bioengineeringPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

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

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Preserving Nanotherapy-Induced Allogeneic Murine Islet Graft Tolerance During Distal Methicillin-Resistant Staphylococcus aureus Skin Infection

Eliminating lifelong immunosuppression can make islet transplantation a durable alternative to insulin therapy for type 1 diabetes. As short-course tolerogenic regimens are developed to replace lifelong immunosuppression, it is critical to test their robustness against perturbations such as hospital-acquired infections, which transplant recipients routinely encounter. Herein, we investigated whether rapamycin-loaded polymersomes (rPS), a subcutaneous nanotherapy that induces long-term murine islet allograft survival, can preserve host defenses in a Methicillin-resistant Staphylococcus aureus (MRSA) skin infection, and whether the resulting immune response from this distal infection relative to graft location erodes rPS-induced tolerance. rPS maintained neutrophil antibacterial activity despite inhibiting oxidative mechanisms in vitro, and rPS-treated mice demonstrated normal recovery following MRSA challenge. However, MRSA skin infection caused a decline of ~43% in recipient survival for rPS-treated mice, despite surviving recipients retaining glucose-stimulated insulin responsiveness. This erosion of allograft acceptance coincided with a burst of IL-6 within 24h of the infection followed by elevated TNF-[a] and neutrophilia at 13 days post-infection. Combining rPS with a single dose of methylprednisolone reduced IL-6 and TNF- without altering neutrophilia, nearly doubling survival among infected recipients to 80%. Our findings demonstrate that partial dampening of inflammatory insults can restore superior islet allograft acceptance following perioperative infection.

bioengineering↗
bioRxiv2026

Extensional Flow Microfluidics Reveal Size-Dependent Core-Shell Mechanical Heterogeneity in Pancreatic Tumor Spheroids

Understanding the mechanical behavior of cancer cell aggregates is important for investigating metastatic potential and developing effective therapeutics. Tumor cell clusters are major contributors to metastasis and can undergo extreme deformation to migrate through capillaries smaller than the cluster size, suggesting complex multicellular mechanics. Despite their clinical significance, the mechanics of multicellular spheroids remain underexplored. In this study, the mechanical behavior of spheroids formed from the pancreatic cancer cell line PANC-1 was investigated using a cross-slot microfluidic device. Suspended spheroids from two size ranges were stretched in non-contact extensional flow (small spheroids mostly 40 < D0 < 75 m, large spheroids mostly 90 < D0 < 145 m). Digital image correlation analyses of intra-spheroid strains revealed strong deformation differences for small and large spheroids while analyses based on 2D projected contours of the spheroid shape did not show trends. Smaller spheroids deformed relatively homogeneously and exhibited significant overall stretching. In contrast, larger spheroids displayed heterogeneous deformation characterized by a stiff central core and a deformable outer shell. To investigate the origin of these mechanical differences, cellular morphology and the expression of stiffness-associated proteins, including vimentin and E-cadherin, were assessed from confocal microscopy. Larger spheroids display significantly lower nuclear sphericity than small spheroids, indicating more cellular packing with increasing spheroid size. Additionally, vimentin expression was concentrated in the outer shell of large spheroids, whereas small spheroids exhibit vimentin expression throughout the aggregate. Thus individual cell morphology and cytoskeletal networks seemed to contribute to the core-shell biomechanical behavior of the large spheroids and lack thereof for small spheroids. Taken together, this study suggests that as spheroids grow, they transition from an irregular and disorganized cluster at low cell numbers to a coordinated and hierarchical structure at sufficient cell numbers that corresponds to diameters {gtrsim} 100 m.

bioengineering↗
bioRxiv2026

Material context and TGF-β blockade timing determine retained matrix in patient-derived bioprinted keratoconus stroma

Stromal repair requires newly formed matrix to persist while the contractile response to TGF-{beta} is controlled. We developed patient-derived bioprinted gelatin methacryloyl (GelMA) stroma to connect material-dependent cell responses with matrix retention after timed receptor blockade. In constructs from six keratoconus (KC) and six normal donors, KC cells retained disease-associated transcription while secreting procollagen I and lumican at normal-donor levels per unit DNA. Comparisons with two-dimensional culture and matched cast GelMA showed that the three-dimensional environment increased matrix induction and lowered resting contractile expression. Printing added a stromal identity response that persisted as retained lumican. Delayed blockade after a TGF-{beta}1 pulse preserved matrix transcription and late secretion. Eleven days after withdrawal, this finite course left more native collagen and lower contractile-gene expression than vehicle in 9/12 donors. Across the tested blockade regimens, at least one regimen achieved both outcomes at the final endpoint in 11/12 donors, including every KC donor. Culture format changed the preferred regimen. Choosing finite blockade by retained collagen instead of secretion changed the schedule in half the donors; the selected arms contained 22.2% more collagen. Retained collagen and contractile expression therefore provide joint endpoints for designing when TGF-{beta}-pathway courses, including losartan and TGF-{beta}3 treatment for keratoconic and scarred corneas, should start and stop.

bioengineering↗