In this paper, we present the ImmunoDisk, a fully automated sample-to-answer centrifugal microfluidic cartridge, integrating a heterogeneous, wash-free, magnetic- and fluorescent bead-based immunoassay (bound-free phase detection immunoassay/BFPD-IA). The BFPD-IA allows the implementation of a simple fluidic structure, where the assay incubation, bead separation and detection are performed in the same chamber. The system was characterized using a C-reactive protein (CRP) competitive immunoassay. A parametric investigation on air drying of protein-coupled beads for pre-storage at room temperature is presented. The key parameters were buffer composition, drying temperature and duration. A protocol for drying two different types of protein-coupled beads with the same temperature and duration using different drying buffers is presented. The sample-to-answer workflow was demonstrated measuring CRP in 5 µL of human serum, without prior dilution, utilizing only one incubation step, in 20 min turnaround time, in the clinically relevant concentration range of 15-115 mg/L. A reproducibility assessment over three disk batches revealed an average signal coefficient of variation (CV) of 5.8 ± 1.3%. A CRP certified reference material was used for method verification with a concentration CV of 8.6%. Our results encourage future testing of the CRP-ImmunoDisk in clinical studies and its point-of-care implementation in many diagnostic applications.

• Klíčová slova
• bound-free phase, centrifugal microfluidics, immunoassay, inflammation, micro/nanoparticles, point-of-care, reagent storage,
• MeSH
• C-reaktivní protein * MeSH
• imunoanalýza metody   MeSH
• indikátory a reagencie MeSH
• lidé MeSH
• mikrofluidika * MeSH
• reprodukovatelnost výsledků MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• C-reaktivní protein * MeSH
• indikátory a reagencie MeSH

Periodontitis and dental caries are two major bacterially induced, non-communicable diseases that cause the deterioration of oral health, with implications in patients' general health. Early, precise diagnosis and personalized monitoring are essential for the efficient prevention and management of these diseases. Here, we present a disk-shaped microfluidic platform (OralDisk) compatible with chair-side use that enables analysis of non-invasively collected whole saliva samples and molecular-based detection of ten bacteria: seven periodontitis-associated (Aggregatibacter actinomycetemcomitans, Campylobacter rectus, Fusobacterium nucleatum, Prevotella intermedia, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola) and three caries-associated (oral Lactobacilli, Streptococcus mutans, Streptococcus sobrinus). Each OralDisk test required 400 µL of homogenized whole saliva. The automated workflow included bacterial DNA extraction, purification and hydrolysis probe real-time PCR detection of the target pathogens. All reagents were pre-stored within the disk and sample-to-answer processing took < 3 h using a compact, customized processing device. A technical feasibility study (25 OralDisks) was conducted using samples from healthy, periodontitis and caries patients. The comparison of the OralDisk with a lab-based reference method revealed a ~90% agreement amongst targets detected as positive and negative. This shows the OralDisk's potential and suitability for inclusion in larger prospective implementation studies in dental care settings.

• Klíčová slova
• caries, dental practice, oral health, periodontitis, point-of-care diagnostics, saliva diagnostics, treatment monitoring,
• MeSH
• lidé MeSH
• mikrofluidní analytické techniky * MeSH
• orální zdraví * MeSH
• parodontitida * diagnóza   MeSH
• sliny mikrobiologie   MeSH
• zubní kaz * diagnóza   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH