One-step, wash-free, bead-based immunoassay employing bound-free phase detection

Johannsen, Benita; Karpíšek, Michal; Baumgartner, Desirée; Klein, Vanessa; Bostancı, Nagihan; Paust, Nils; Früh, Susanna M.; Zengerle, Roland; Mitsakakis, Konstantinos

doi:10.1016/j.aca.2021.338280

Cited by 16 publications

(17 citation statements)

References 52 publications

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“…The LabDisk has already been shown to be compatible with immunoassays by the running of a basic reaction (without detection) as a proof-of-principle demonstration [ 74 ]. A newly developed and tested bead-based immunoassay with oral biomarkers [ 75 ] will enable an immunoassay disk to be run in the same instrument as the PCR disk. This will increase the interoperability between immunological and microbiological diagnostic outputs using this platform [ 76 ].…”

Section: Resultsmentioning

confidence: 99%

OralDisk: A Chair-Side Compatible Molecular Platform Using Whole Saliva for Monitoring Oral Health at the Dental Practice

et al. 2021

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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.

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Section: Resultsmentioning

confidence: 99%

OralDisk: A Chair-Side Compatible Molecular Platform Using Whole Saliva for Monitoring Oral Health at the Dental Practice

et al. 2021

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“…The novel BFPD immunoassay was presented and evaluated in detail in a previous publication by Johannsen et al [ 46 ]. The following is a brief summary of the procedure for the detection of CRP in human serum.…”

Section: Methodsmentioning

confidence: 99%

“…In order to assess the activity of the dried bead-coupled antibodies and antigens after pre-storage and resuspension, we used a negative control of the CRP assay in a microtiter plate, as described in a previous publication [ 46 ]. This negative control assay includes both types of coupled beads without the native CRP antigen in the sample.…”

Section: Methodsmentioning

confidence: 99%

“…In the context of the state of the art, we present a fully integrated centrifugal microfluidic system, the ImmunoDisk, implementing, for the first time, an immunoassay method that combines a bead-based and wash-free configuration without additional surface treatments or density media, with all reagents pre-stored, and using bound-free phase detection (BFPD). The bound-free phase detection immunoassay (BFPD-IA) is a heterogeneous assay based on ELISA principles and utilizes magnetic and fluorescent beads, the former acting as capture phase and the latter as the detection agent (further information on the BFPD-IA in Johannsen et al [ 46 ]). The simplicity of the BFPD-IA concept allows the realization of assay incubation, bead separation and detection all in a single chamber.…”

Section: Introductionmentioning

confidence: 99%

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ImmunoDisk—A Fully Automated Bead-Based Immunoassay Cartridge with All Reagents Pre-Stored

et al. 2022

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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.

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“…Bead-based immunoassays using fluorescent nanoparticles for detection can ensure multiplexing and render the immunoassay compatible with the TaqMan probe PCR detection during the integration in automated systems. Furthermore, the flexibility and adaptability of such assays with diverse media (blood and saliva in case of our two examined disease cases) is important [19].…”

Section: Bioassaysmentioning

confidence: 99%

One-step, wash-free, bead-based immunoassay employing bound-free phase detection

Cited by 16 publications

References 52 publications

OralDisk: A Chair-Side Compatible Molecular Platform Using Whole Saliva for Monitoring Oral Health at the Dental Practice

OralDisk: A Chair-Side Compatible Molecular Platform Using Whole Saliva for Monitoring Oral Health at the Dental Practice

ImmunoDisk—A Fully Automated Bead-Based Immunoassay Cartridge with All Reagents Pre-Stored

Novel lab-on-a-disk platforms: a powerful tool for molecular fingerprinting of oral and respiratory tract infections

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