A sensitive and compact on-site and real time quantitative detection of foodborne and clinical pathogens by on-chip qPCR Ana João Pereira, Steven Schoonderwoerd, Rodrigo Sergio Wiederkehr, Daniele Chieffi, Vincenzina Fusco, Gabriela Vollet Marson Talanta Open, 2026 • On-chip qPCR of Staphylococcus aureus can be completed under 20 minutes. • The optimized and validated assay consisted of KAPA2G polymerase with EvaGreen dye. • DoE revealed influential factors and optimized conditions for on-chip qPCR. • Limit of detection is four genome equivalents per reaction (1.8 µL). • Low-cost recipe achieved high PCR efficiency, sensitivity and specificity. Herein we report the quantitative detection of pathogenic bacteria by an on-chip qPCR previously developed for COVID-19 detection, using Staphylococcus aureus as pilot microorganism. A conventional qPCR assay for the rapid and sensitive detection of Staphylococcus aureus harbouring the enterotoxin gene cluster ( egc ) was transferred to a microfluidic platform. Patterned silicon substrates were used as reaction vessels during on-chip qPCR. Reagent optimization was achieved through Design of Experiments (DoE), using multifactorial analysis with fractional factorial and central composite rotatable designs. On-chip thermal cycling and optical inspection were achieved using a compact reader providing real-time monitoring and accurate quantification. Experiments were performed to evaluate optimal assay efficiency, sensitivity, specificity, practicability, and robustness. The assay was tested for probe-based or intercalating dye-based amplification systems as those are fastest and most sensitive. Seven relevant components in the reaction mixture were screened through 16 amplification runs, where cost, speed and amplification quality were assessed. The screening identified the intercalating dye-based system as most promising, with polymerase and primer concentrations as the most influential factors. The following optimization design led to bacterial screening with template concentrations ranging from 4 to 40,000 copies in 1.8 µL of mixture per reaction under 19 minutes, compared to 10 µL of mixture per reaction under 40 minutes of run time using the conventional real time PCR platform. The workflow introduced here simplifies the transfer of qPCR recipes to microfluidic devices targeting faster results when compared with the current gold standard benchtop methods on pathogen detection in real time at on-site locations.
A capillary-driven microfluidic device for performing spatial multiplex PCR Rodrigo S. Wiederkehr, Elisabeth Marchal, Maarten Fauvart, Tomas Forceville, Ahmed Taher, Tim Steylaerts, YoungJae Choe, Hans Dusar, Silvia Lenci, Eleni Siouti, Vassiliki T. Potsika, Evangelos Andreakos, Tim Stakenborg Biomedical Microdevices, 2025
Molecular detection of SARS-COV-2 in exhaled breath at the point-of-need Tim Stakenborg, Joren Raymenants, Ahmed Taher, Elisabeth Marchal, Bert Verbruggen, Sophie Roth, Ben Jones, Abdul Yurt, Wout Duthoo, Klaas Bombeke, Maarten Fauvart, Julien Verplanken, Rodrigo S. Wiederkehr, Aurelie Humbert, Chi Dang, Evi Vlassaks, Alejandra L. Jáuregui Uribe, Zhenxiang Luo, Chengxun Liu, Kirill Zinoviev, Riet Labie, Aduen Darriba Frederiks, Jelle Saldien, Kris Covens, Pieter Berden, Bert Schreurs, Joost Van Duppen, Rabea Hanifa, Megane Beuscart, Van Pham, Erik Emmen, Annelien Dewagtere, Ziduo Lin, Marco Peca, Youssef El Jerrari, Chinmay Nawghane, Chad Arnett, Andy Lambrechts, Paru Deshpande, Katrien Lagrou, Paul De Munter, Emmanuel André, Nik Van den Wijngaert, Peter Peumans Biosensors and Bioelectronics, 2022 The SARS-CoV-2 pandemic has highlighted the need for improved technologies to help control the spread of contagious pathogens. While rapid point-of-need testing plays a key role in strategies to rapidly identify and isolate infectious patients, current test approaches have significant shortcomings related to assay limitations and sample type. Direct quantification of viral shedding in exhaled particles may offer a better rapid testing approach, since SARS-CoV-2 is believed to spread mainly by aerosols. It assesses contagiousness directly, the sample is easy and comfortable to obtain, sampling can be standardized, and the limited sample volume lends itself to a fast and sensitive analysis. In view of these benefits, we developed and tested an approach where exhaled particles are efficiently sampled using inertial impaction in a micromachined silicon chip, followed by an RT-qPCR molecular assay to detect SARS-CoV-2 shedding. Our portable, silicon impactor allowed for the efficient capture (>85%) of respiratory particles down to 300 nm without the need for additional equipment. We demonstrate using both conventional off-chip and in-situ PCR directly on the silicon chip that sampling subjects' breath in less than a minute yields sufficient viral RNA to detect infections as early as standard sampling methods. A longitudinal study revealed clear differences in the temporal dynamics of viral load for nasopharyngeal swab, saliva, breath, and antigen tests. Overall, after an infection, the breath-based test remains positive during the first week but is the first to consistently report a negative result, putatively signalling the end of contagiousness and further emphasizing the potential of this tool to help manage the spread of airborne respiratory infections.
Amplification Efficiency and Template Accessibility as Distinct Causes of Rain in Digital PCR: Monte Carlo Modeling and Experimental Validation Pieter Berden, Rodrigo S. Wiederkehr, Liesbet Lagae, Jan Michiels, Tim Stakenborg, Maarten Fauvart, Willem Van Roy Analytical Chemistry, 2022 Partitions in digital PCR (dPCR) assays do not reach the detection threshold at the same time. This heterogeneity in amplification results in intermediate endpoint fluorescence values (i.e., rain) and misclassification of partitions, which has a major impact on the accuracy of nucleic acid quantification. Rain most often results from a reduced amplification efficiency or template inaccessibility; however, exactly how these contribute to rain has not been described. We developed and experimentally validated an analytical model that mechanistically explains the relationship between amplification efficiency, template accessibility, and rain. Using Monte Carlo simulations, we show that a reduced amplification efficiency leads to broader threshold cycle (Ct) distributions that can be fitted using a log-normal probability distribution. From the fit parameters, the amplification efficiency can be calculated. Template inaccessibility, on the other hand, leads to a different rain pattern, in which a distinct exponential tail in the Ct distribution can be observed. Using our model, it is possible to determine if the amplification efficiency, template accessibility, or another source is the main contributor of rain in dPCR assays. We envision that this model will facilitate and speed up dPCR assay optimization and provide an indication for the accuracy of the assay.
Model-based classification of rain in digital PCR as a benchmark for assay reliability Microtas 2021 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences, 2021
Silicon µPCR Chip for Forensic STR Profiling with Hybeacon Probe Melting Curves Senne Cornelis, Olivier Tytgat, Maarten Fauvart, Yannick Gansemans, Ann-Sophie Vander Plaetsen, Rodrigo S. Wiederkehr, Dieter Deforce, Filip Van Nieuwerburgh, Tim Stakenborg Scientific Reports, 2019 The demand to perform forensic DNA profiling outside of centralized laboratories and on the crime scene is increasing. Several criminal investigations would benefit tremendously from having DNA based information available in the first hours rather than days or weeks. However, due to the complexity and time-consuming nature of standard DNA fingerprinting methods, rapid and automated analyses are hard to achieve. We here demonstrate the implementation of an alternative DNA fingerprinting method in a single microchip. By combining PCR amplification and HyBeacon melting assays in a silicon Lab-on-a-chip (LoC), a significant step towards rapid on-site DNA fingerprinting is taken. The small form factor of a LoC reduces reagent consumption and increases portability. Additional miniaturization is achieved through an integrated heating element covering 24 parallel micro-reactors with a reaction volume of 0.14 µl each. The high level of parallelization allows the simultaneous analysis of 4 short tandem repeat (STR) loci and the amelogenin gender marker commonly included in forensic DNA analysis. A reference and crime scene sample can be analyzed simultaneously for direct comparison. Importantly, by using industry-standard semiconductor manufacturing processes, mass manufacturability can be guaranteed. Following assay design and optimization, complete 5-loci profiles could be robustly generated on-chip that are on par with those obtained using conventional benchtop real-time PCR thermal cyclers. Together, our results are an important step towards the development of commercial, mass-produced, portable devices for on-site testing in forensic DNA analysis.
Development and validation of a glass-silicon microdroplet-based system to measure sulfite concentrations in beverages Yannick Vervoort, Rodrigo Sergio Wiederkehr, Michiel Smets, Maarten Fauvart, Tim Stakenborg, Gabrielle Woronoff, Liesbet Lagae, Kevin J. Verstrepen Analytical and Bioanalytical Chemistry, 2019 Sulfite is often added to beverages as an antioxidant and antimicrobial agent. In fermented beverages, sulfite is also naturally produced by yeast cells. However, sulfite causes adverse health effects in asthmatic patients and accurate measurement of the sulfite concentration is therefore very important. Current sulfite analysis methods are time- and reagent-consuming and often require costly equipment. Here, we present a system allowing sensitive, ultralow-volume sulfite measurements based on a reusable glass-silicon microdroplet platform on which microdroplet generation, addition of enzymes through chemical-induced emulsion destabilization and pillar-induced droplet merging, emulsion restabilization, droplet incubation, and fluorescence measurements are integrated. In a first step, we developed and verified a fluorescence-based enzymatic assay for sulfite by measuring its analytical performance (LOD, LOQ, the dynamic working range, and the influence of salts, colorant, and sugars) and comparing fluorescent microplate readouts of fermentation samples with standard colorimetric measurements using the 5,5'-dithiobis-(2-nitrobenzoic acid) assay of the standard Gallery Plus Beermaster analysis platform. Next, samples were analyzed on the microdroplet platform, which also showed good correlation with the standard colorimetric analysis. Although the presented platform does not allow stable reinjection of droplets due to the presence of a tight array of micropillars at the fluidics entrances to prevent channel clogging by dust, removing the pillars, and integrating miniaturized pumps and optics in a future design would allow to use this platform for high-throughput, automated, and portable screening of microbes, plant, or mammalian cells. Graphical abstract ᅟ.
A droplet microfluidics platform for scalable and high-throughput isolation of antibiotic-producing microbes 23rd International Conference on Miniaturized Systems for Chemistry and Life Sciences Microtas 2019, 2019
Multiplex STR amplification sensitivity in a silicon microchip Senne Cornelis, Maarten Fauvart, Yannick Gansemans, Ann-Sophie Vander Plaetsen, Frederik Colle, Rodrigo S. Wiederkehr, Dieter Deforce, Tim Stakenborg, Filip Van Nieuwerburgh Scientific Reports, 2018 The demand for solutions to perform forensic DNA profiling outside of centralized laboratories is increasing. We here demonstrate highly sensitive STR amplification using a silicon micro-PCR (µPCR) chip. Exploiting industry-standard semiconductor manufacturing processes, a device was fabricated that features a small form factor thanks to an integrated heating element covering three parallel micro-reactors with a reaction volume of 0.5 µl each. Diluted reference DNA samples (1 ng-31 pg) were amplified on the µPCR chip using the forensically validated AmpFISTR Identifier Plus kit, followed by conventional capillary electrophoresis. Complete STR profiles were generated with input DNA quantities down to 62 pg. Occasional allelic dropouts were observed from 31 pg downward. On-chip STR profiles were compared with those of identical samples amplified using a conventional thermal cycler for direct comparison of amplification sensitivity in a forensic setting. The observed sensitivity was in line with kit specifications for both µPCR and conventional PCR. Finally, a rapid amplification protocol was developed. Complete STR profiles could be generated in less than 17 minutes from as little as 125 pg template DNA. Together, our results are an important step towards the development of commercial, mass-produced, relatively cheap, handheld devices for on-site testing in forensic DNA analysis.
Beer on a chip: Identification of superior industrial yeasts using droplet microfluidics 20th International Conference on Miniaturized Systems for Chemistry and Life Sciences Microtas 2016, 2016
Detection of multiple single nucleotide polymorphisms on a single microfluidic chip 20th International Conference on Miniaturized Systems for Chemistry and Life Sciences Microtas 2016, 2016
Digital droplet PCR on chip for quantitative assessment of microRNAs 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences Microtas 2014, 2014
On-chip multiplex PCR amplification directly from whole blood 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences Microtas 2013, 2013
Multiplex ligation-dependent probe amplification (MLPA) on-chip 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences Microtas 2013, 2013
