Detection and enumeration of bacteria by pump-free digital droplet assays

Abstract: Bacteria are present in all sections of our life. However, in order to assess and control health threats, it is necessary to detect and enumerate critical bacterial species in order to prevent bacteria outbreak, diagnose bacterial infections, to provide a proper treatment and further to avoid antibiotic abuses. Current techniques for detection and enumeration of bacteria are time-consuming, labor-intensive, often lack of precision, or require additional calibration strategies. Although digital droplet assays provide an opportunity for absolute quantification of bacteria and can reduce the time-to-result, the current droplet microfluidic setups for bacterial enumeration have a low degree of integration and parallelization, comprise complicated manual handling steps and need external interfaces such as connecting tubings. Therefore, there is a need to fill these gaps.
This thesis presents the implementation approaches of three bacterial detection assays that use pump-free step emulsification cartridges for digital detection and quantification of bacteria. The first implementation was the integration of a resazurin reduction assay on a newly developed gravity-driven step emulsification device for universal bacterial detection and enumeration being applied to a digital antibiotic susceptibility test. The four-chamber gravity-driven step emulsification device allowed encapsulation of bacteria suspension in 2000 of 2 nl droplets into each chamber. The manual workload for droplet generation involved merely sample preparation, loading the sample mixture into the sample inlet of the device, and subsequently orienting the device vertically for gravity-driven emulsification. Thereby, no additional pumping system was required. In the digital detection and enumeration assay, the resazurin was reduced to fluorescent resorufin in the presence of viable bacteria and the quantification of bacteria was reduced from 18–24 hours down to 5 hours. Decimal dilution series showed a high degree of linearity of the assay for the bacterial species: Escherichia coli (R2=0.9985), Staphylococcus aureus (R2=0.9998), and Enterococcus faecalis (R2=0.9992).
The second implementation was the integration of a generic enzymatic substrate method by using resorufin β-D-glucuronide on centrifugal step emulsification cartridge (DropChip) for detection and enumeration of Escherichia coli. A two-chamber DropChip generated 35600 of
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0.42 nl droplets in each chamber with the aid of a standard laboratory mini-centrifuge. Methyl-β-D-glucuronide was used to induce the production of β-D-glucuronidase in Escherichia coli so that resorufin β-D-glucuronide was hydrolyzed and released a fluorophore for signal detection. The digital enumeration of Escherichia coli can be achieved in 4.5 hours and displayed high correlations (R2=0.9999) with the standard plate count.
The third implementation was the integration of a one-pot wash-free fluorescence in situ hybridization assay on a centrifugal step emulsification cartridge (LabDisk) for universal but gene specific bacterial detection and enumeration. A ten-chamber LabDisk generated 47600 of 0.21 nl droplets per chamber with the aid of a customized centrifuge (LabDisk Player). The method offers minimal manual workload – it requires only mixing of the sample with the reagents and loading it into a cartridge for partitioning of the bacteria into droplets for in situ permeabilization, hybridization and signal detection. Optimized LNA/DNA molecular beacons targeted the universal region of bacterial 16S ribosomal RNA and allowed visualization of bacteria at single cell level. The assay was characterized with Escherichia coli, Klebsiella pneumonia, and Proteus mirabilis with single-cell sensitivity and a linearity of R2=0.9760 for all data points including variation of the three tested bacteria models. The total time-to-result of digital enumeration of bacteria was around 1.5 hours.
In conclusion, this thesis provides easy-to-use pump-free digital droplet assay solutions for same day timely bacteria detection and enumeration with minimal manual handling steps and simple workflow. The presented three methods are capable to pave the way for further implementation in point-of-care detection systems in the future

Angaben zum Objekt

• Standort: Deutsche Nationalbibliothek Frankfurt am Main
• Umfang: Online-Ressource
• Sprache: Englisch
• Anmerkungen: Universität Freiburg, Dissertation, 2022

Klassifikation und Themen

• Schlagwort: Bakterien ; Mikroorganismus ; Nachweis ; Biochemische Analyse

Beteiligte, Orts- und Zeitangaben

• Ereignis: Veröffentlichung
• (wo): Freiburg
• (wer): Universität
• (wann): 2022
• Urheber: Kao, Yu-Ting
• Beteiligte Personen und Organisationen: Zengerle, Roland ; Zengerle, Roland ; Garstecki, Piotr ; Albert-Ludwigs-Universität Freiburg. Fakultät für Angewandte Wissenschaften

Weitere Informationen

• DOI: 10.6094/UNIFR/229643
• URN: urn:nbn:de:bsz:25-freidok-2296434
• Rechteinformation: Open Access; Der Zugriff auf das Objekt ist unbeschränkt möglich.
• Letzte Aktualisierung: 25.03.2025, 13:55 MEZ