The project “µ‑Viroscreen – a new microfluidic approach for the quantification of infectious viruses” was carried out from 1 July 2021 to 31 December 2023 in cooperation between the Institute for Bioprocess and Analysis Measurement Technology (iba) in Heiligenstadt and the Technical University of Middle Hesse (THM) in Giessen, under the auspices of the German Federal Ministry of Education and Research (BMWK) through the Advanced Innovation Fund (AiF). The research consortium DECHEMA coordinated the effort, with the two institutes sharing responsibilities: iba focused on the design, fabrication and automation of the microfluidic platform, while THM provided expertise in virology, flow cytometry and quantitative PCR (qPCR) and handled the biological validation of the assays.

The core scientific objective was to transfer a conventional virus‑quantification assay into an automated microfluidic system and to demonstrate that this approach can reduce the time required for detecting infectious baculoviruses (BV) compared with standard methods such as TCID50. Two baculovirus genotypes were used: a wild‑type AcMNPV and a GFP‑expressing variant (AcMNPV‑GFP). Sf‑9 insect cells were infected in serum‑free medium, and viral DNA was extracted for qPCR calibration. The qPCR assay achieved a ten‑fold increase in virus titer from 1.0 × 10⁶ to 1.0 × 10⁷ copies mL⁻¹, with a linear calibration range spanning 1.1 × 10⁷ to 1.1 × 10² genome copies mL⁻¹. The correlation coefficient was R² = 0.995 and the amplification efficiency was 1.04, indicating excellent reproducibility; replicate variation was below 0.6 CT (less than 3 %). These data provided a robust reference for the microfluidic measurements.

For flow cytometry, the team optimized detection of infected cells by measuring GFP fluorescence for the GFP‑virus and by staining the surface protein gp64 with an anti‑gp64 antibody for the wild‑type virus. The assay required overnight incubation for GFP detection, whereas gp64 staining could be detected within minutes. A linear calibration curve was established for both detection modes. Attempts to quantify viral mRNA by fluorescence in‑situ hybridization were not reproducible and proved too costly and time‑consuming, so this approach was not pursued further.

The microfluidic platform integrated modular fluidic microsystems (FMS) for droplet generation, virus injection, incubation, and downstream analysis. Droplet‑in‑droplet design allowed the transport of fluorescence‑labelled compartments without surfactants, enabling direct coupling to a flow cytometer. The system achieved detection of viral surface proteins after only 5 hours of incubation, a substantial reduction compared with conventional TCID50 assays that require several days. GFP‑based detection in the microfluidic mode required up to 20 hours, matching the time needed in the off‑line assay. Importantly, injection tests confirmed that the platform prevented cross‑contamination between droplets, ensuring assay robustness.

Overall, the project demonstrated that an automated microfluidic workflow can provide rapid, quantitative detection of infectious baculoviruses with performance metrics comparable to established qPCR and flow cytometry methods. The collaboration between iba and THM, supported by DECHEMA and funded by BMWK, successfully achieved the stated goal of developing a µ‑Viroscreen platform that shortens virus quantification timelines and enhances process analytical technology for biopharmaceutical production.