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. 2024 May;416(13):3107-3115.
doi: 10.1007/s00216-024-05271-3. Epub 2024 Apr 9.

Sample-to-answer lateral flow assay with integrated plasma separation and NT-proBNP detection

Dan Strohmaier-Nguyen  1 , Carina Horn  2 , Antje J Baeumner  3
Affiliations

Sample-to-answer lateral flow assay with integrated plasma separation and NT-proBNP detection

Dan Strohmaier-Nguyen et al. Anal Bioanal Chem. 2024 May.

Abstract

Through enabling whole blood detection in point-of-care testing (POCT), sedimentation-based plasma separation promises to enhance the functionality and extend the application range of lateral flow assays (LFAs). To streamline the entire process from the introduction of the blood sample to the generation of quantitative immune-fluorescence results, we combined a simple plasma separation technique, an immunoreaction, and a micropump-driven external suction control system in a polymer channel-based LFA. Our primary objective was to eliminate the reliance on sample-absorbing separation membranes, the use of active separation forces commonly found in POCT, and ultimately allowing finger prick testing. Combining the principle of agglutination of red blood cells with an on-device sedimentation-based separation, our device allows for the efficient and fast separation of plasma from a 25-µL blood volume within a mere 10 min and overcomes limitations such as clogging, analyte adsorption, and blood pre-dilution. To simplify this process, we stored the agglutination agent in a dried state on the test and incorporated a filter trench to initiate sedimentation-based separation. The separated plasma was then moved to the integrated mixing area, initiating the immunoreaction by rehydration of probe-specific fluorophore-conjugated antibodies. The biotinylated immune complex was subsequently trapped in the streptavidin-rich detection zone and quantitatively analyzed using a fluorescence microscope. Normalized to the centrifugation-based separation, our device demonstrated high separation efficiency of 96% and a yield of 7.23 µL (= 72%). Furthermore, we elaborate on its user-friendly nature and demonstrate its proof-of-concept through an all-dried ready-to-go NT-proBNP lateral flow immunoassay with clinical blood samples.

Keywords: Blood plasma separation; Lateral flow assay; Point-of-care diagnostics; Sample-to-answer; Sedimentation.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Antje J. Baeumner is editor of this journal but was not involved in the peer review of this article.

Figures

Fig. 1
Fig. 1
Schematic (not to scale) of the lateral flow channel assay with integrated plasma separation. After introducing the sample to the inlet (1), the sample is transported to the filter trench by the fluid control system. In the filter trench, the RBCs are aggregated and separated from the plasma through sedimentation (2). The separated plasma is then moved from the plasma separation module towards the reaction zone, reconstituting the probe antibodies and initiating the immunoreaction. In the detection zone, streptavidin is immobilized on the bottom of the channel for capturing the biotinylated sandwich complex (3). Adapted from “PDMS microfluidic chip fabrication”, by BioRender.com (2023). Retrieved from https://app.biorender.com/biorender-templates
Fig. 2
Fig. 2
Optimization of the separated plasma volume (a) and optimization of the plasma purity (b) with respect to the trench depth and the time for separation. “c” represents the plasma separation through centrifugation. The lateral flow channel was loaded with 25 µL of whole blood (hematocrit, 44%), and the purity as well as the volume of plasma obtained was measured. Error bars represent the standard deviation (n = 4)
Fig. 3
Fig. 3
Plot of fluorescence intensity against logarithm of antigen concentration. Fluorescent read-out of the analyte in 25 µL whole blood with logistic fit (black line) and using the integrated plasma separation with logistic fit (blue line). Standard deviations were calculated based on four parallel measurements on four different LFAs, while outliers were removed after Q-test (confidence interval 95%). Error bars represent mean values ± 1σ (n ≥ 4)
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