A novel multianalyte lateral flow immunoassay for the simultaneous detection of protein and nucleic acid biomarkers

Abstract: Point-of-care (POC) testing has become an invaluable tool in diagnostics by offering rapid turnaround times and simple sample-to-answer workflows that even can be performed by non experts. The most used type of POC test for medical diagnostics is the lateral flow test (such as the pregnancy test and COVID-19 rapid antigen test). The COVID-19 pandemic, in particular, has contributed to the fact that the basic understanding, acceptance, and experience in handling these paper-based rapid tests has reached the broader public.
Innovations in paper-based POC diagnostics have provoked the interest in combining nucleic acid amplification tests (NAATs) with lateral flow assays. In this regard, the development of rapid isothermal nucleic acid amplification methods, which erase the need for thermal cycling, was a key innovation towards paper-based POC NAATs. This development opens new possibilities – especially for infectious diseases where a rapid identification of pathogens and target-oriented therapy is of uttermost importance.
The need to detect multiple analytes from a single sample, known as multianalyte detection, has grown in importance in recent years. The regulation of disease progression involves highly interactive processes influenced by various classes of biomolecules (such as proteins and nucleic acids). Therefore, a more comprehensive analysis of biomarkers (including different biomolecule classes) from a single sample is highly desirable and can greatly improve medical diagnostics. Wound infections, for example, are a substantial problem for the patient’s health and healthcare systems. Monitoring of wounds and differentiation between active and inactive infections is enabled by the simultaneous detection of local host immune response biomarkers and pathogens via nucleic acid analysis.
Currently, there are no paper-based biosensors that (i) enable the simultaneous detection of protein and nucleic acid biomarkers (ii) from a single sample, (iii) use the same reaction conditions, and (iv) meet the requirements for medical POC diagnostics. Therefore, it is of uttermost importance to develop new paper-based multianalyte biosensors that address this need and fulfill these requirements.
The overall aim of this thesis was to develop a novel paper-based multianalyte biosensor that enables the simultaneous detection of target proteins and nucleic acids from a single sample. To achieve this objective, a Multianalyte-Assay was invented, which encodes the presence of nucleic acids into a protein-based biorecognition format (nucleic acid-to-protein transformation) and subsequently detects both biomolecule classes (nucleic acids and proteins) via immunoassay (such as a lateral flow immunoassay). Wound infection was chosen as a prominent example, where a rapid and simple detection of multiple biomolecule classes is very beneficial. To demonstrate the feasibility of the multianalyte lateral flow immunoassay, genomic DNA (gDNA) of the opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa) and the proinflammatory cytokine interleukin-6 (IL-6) were chosen as model markers.
First, a nucleic acid lateral flow immunoassay was developed for the direct detection of isothermally amplified and labeled pathogen DNA combined with fast sample preparation. The streamlined protocol was evaluated using human wound exudate spiked with P. aeruginosa. The paper-based approach only requires three simple hands-on steps and has a turn-around time of less than 30 min. A detection limit of 2.1 × 105 CFU per mL wound fluid was achieved and an internal amplification control that excludes false negative results was implemented.
Next, the pathogen detection was combined with the detection of the local inflammation marker IL-6. Both biomarkers, P. aeruginosa gDNA and IL-6, were added simultaneously to the amplification reaction, which amplified and labeled the gDNA at 37 °C. Subsequently, both biomarkers were detected simultaneously via lateral flow immunoassay. The multianalyte lateral flow immunoassay achieved a turn-around time of 35 min and detection limits of 4 ng/mL for IL-6 and 70 copies/reaction for P. aeruginosa gDNA.
Thus, this achievement is a significant step towards the vision of a of a paper-based POC multianalyte biosensor for the simultaneous detection of proteins and nucleic acids from a single sample and has tremendous potential to improve wound management at the POC. This approach can also be transferred to other fields of application, such as the stratification of acute respiratory infections. In future, this could enable faster and targeted treatments

Object Details

• Location: Deutsche Nationalbibliothek Frankfurt am Main
• Extent: Online-Ressource
• Language: Englisch
• Notes: Universität Freiburg, Dissertation, 2024

Classification and Topics

• Keyword: Biosensor

Contributors, Places and Time

• Event: Veröffentlichung
• (where): Freiburg
• (who): Universität
• (when): 2024
• Creator: Klebes, Anna
• Contributor: Stetten, Felix von ; Stetten, Felix von ; Rühe, Jürgen ; Albert-Ludwigs-Universität Freiburg. Fakultät für Angewandte Wissenschaften

Further information

• DOI: 10.6094/UNIFR/255812
• URN: urn:nbn:de:bsz:25-freidok-2558129
• Rights: Open Access; Der Zugriff auf das Objekt ist unbeschränkt möglich.
• Last update: 25.03.2025, 1:47 PM CET