Biology subjects

Ejendal, K. F. K.

Publications and source records attributed to Ejendal, K. F. K..

3 recordsLinked to original sources

bioRxiv2025

Antibody-Initiated Loop-Mediated Isothermal Nucleic Acid Amplification (ai-LAMP) as a New Biosensor for Antigen Detection

Highly sensitive protein detection is critical in research and healthcare diagnostics but remains limited to resource intensive environments. In contrast, lateral flow immunoassay (LFIA)-based diagnostics are popular point-of-care tests due to their simplicity and user-friendly format but lack sensitivity for detecting the low concentrations of proteins that are present early in infections and among asymptomatic individuals. To overcome these limitations, we developed a novel biosensor platform utilizing antibody-initiated loop-mediated isothermal amplification (ai-LAMP) with an LFIA readout for protein detection. This platform integrates protein-specific antibody-antigen binding with the robust signal amplification of LAMP. By conjugating pairs of antibodies to overlapping DNA strands, the presence of a target protein brings the DNA strands into proximity, completing a DNA target to directly initiate the LAMP reaction without any additional ligation step. This approach facilitates the rapid detection of low concentration proteins with a clear visual readout and can be performed in 3 steps from sample to answer. Using ai-LAMP we detected HIV-1 p24 on commercially available LFIAs at a limit of detection (LOD) of 20 fM (0.53 pg/mL), demonstrating 46x improvement over existing HIV p24 LFIAs. The use of ai-LAMP eliminates the need for sophisticated laboratory equipment to detect protein targets in low concentrations, paving a new way for rapid and accessible biomarker detection in clinical and research settings. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/678293v2_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@5b587forg.highwire.dtl.DTLVardef@10f3587org.highwire.dtl.DTLVardef@1833eeborg.highwire.dtl.DTLVardef@197d1bf_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Mechanisms of Alpha-Synuclein Seeded Aggregation in Neurons Revealed by Fluorescence Lifetime Imaging

1The brains of Parkinsons disease (PD) patients are characterized by the presence of Lewy body inclusions enriched with fibrillar forms of the presynaptic protein alpha-synuclein (aSyn). Despite related evidence that Lewy pathology spreads across different brain regions as the disease progresses, the underlying mechanism hence the fundamental cause of PD progression is unknown. The propagation of aSyn pathology is thought to potentially occur through the release of aSyn aggregates from diseased neurons, their uptake by neighboring healthy neurons via endocytosis, and subsequent seeding of native aSyn aggregation in the cytosol. A critical aspect of this process is believed to involve the escape of internalized ag-gregates from the endolysosomal compartment, though direct evidence of this mechanism in cultured neuron models remains lacking. In this study, we utilize a custom-built, time-gated fluorescence lifetime imaging microscope (FLIM) to investigate the progression of seeded ag-gregation over time in live cortical neurons. By establishing fluorescence lifetime sensitivity to aSyn aggregation level, we are able to monitor the proteins aggregation state. Through a FLIM analysis of neurons expressing aSyn-mVenus and exposed to aSyn preformed fibrils labeled with the acid-responsive dye pHrodo, we reveal the proteins aggregation state in both the cytosol and the endolysosomal compartment. The results indicate that aSyn seeds undergo partial disassembly prior to escaping the endocytic pathway, and that this escape is closely linked to the aggregation of cytosolic aSyn. In certain neurons, monomeric aSyn is found to translocate from the cytosol into the endolysosomal compartment, where it appar-ently forms aggregates in proximity to retained seeds. Additional analyses reveals zones of neuritic aSyn aggregates that overlaps with regions of microtubule disruption. Collectively, these findings enhance our understanding of aSyn pathology propagation in PD and other synucleinopathies, motivate additional experiments along these lines, and offer a path to guide the development of disease-modifying therapies.

neuroscience↗

bioRxiv2019

Microfluidic Rapid and Autonomous Analytical Device (microRAAD) to Detect HIV from Whole Blood Samples

Early Human Immunodeficiency Virus (HIV) testing is critical to preventing transmission and providing treatment to HIV-positive individuals, yet an estimated 30% of HIV-positive individuals do not know their status because of barriers to early diagnosis. Readily accessible, highly sensitive, and rapid diagnostic tests would enable patients prompt treatment with anti-retroviral therapies and reduce transmission. However, existing HIV diagnostic technologies either do not detect early stages of infection or require multiple days of laboratory processing, delaying notification of patients status.\n\nMolecular techniques that amplify HIV RNA can detect the earliest stages of infection, within 8-10 days after transmission. However, most of these molecular assays require cold-chain storage of reagents, significant sample preparation, and extensive laboratory infrastructure. To achieve early detection, we developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay with a limit of detection of 10 HIV-1 RNA copies visualized by eye using a lateral flow immunoassay. To demonstrate automated sample-to-answer detection of HIV, we incorporate dried amplification reagents and wax valves in low-cost substrates with resistive heating elements and circuitry. By combining controlled heating with papers capillary flow, our assembled device automatically isolates viral particles from human blood samples, amplifies HIV-1 RNA, and transports products to a detection zone. We determine that as few as 105 HIV-1 viral particles can be separated from whole blood, amplified, and visually detected within 90 minutes of sample addition into our Microfluidic Rapid and Autonomous Analysis Device (microRAAD). The low-cost and automated attributes of microRAAD demonstrate its utility as a point-of-care testing platform.

bioengineering↗