Malaria diagnosis and prognosis with an electrokinetic-driven aptamer-based lateral flow assay.

Informazioni relative al progetto

MalDiProT

ID dell’accordo di sovvenzione: 101150621

DOI

10.3030/101150621

Data della firma CE 17 Aprile 2024

Data di avvio 1 Ottobre 2024

Data di completamento 30 Settembre 2026

Finanziato da

• Marie Skłodowska-Curie Actions (MSCA)

Costo totale

Nessun dato

Contributo UE

€ 187 858,56

Coordinato da

FUNDACION PRIVADA INSTITUTO DE SALUD GLOBAL BARCELONA
Spain

Periodic Reporting for period 1 - MalDiProT (Malaria diagnosis and prognosis with an electrokinetic-driven aptamer-based lateral flow assay.)

Periodo di rendicontazione: 2024-10-01 al 2025-09-30

Sintesi del contesto e degli obiettivi generali del progetto

Malaria remains one of the world’s most persistent infectious diseases, causing hundreds of thousands of deaths every year and placing nearly half of the global population at risk. Although the disease is most common in sub-Saharan Africa, Europe also faces increasing numbers of imported malaria cases due to international travel and migration. Early diagnosis and rapid identification of patients at risk of developing severe symptoms are crucial for reducing complications and saving lives. However, current diagnostic tools have significant limitations: the gold-standard method, microscopy, requires trained personnel and specialized laboratory equipment, while existing rapid diagnostic tests used outside the laboratory setting are primarily qualitative and fail to detect infections with low parasite levels or provide information on disease severity. As a result, many patients, especially travellers who lack natural immunity, are not identified early enough to prevent life-threatening complications.

This project addresses these challenges by developing a new generation of portable, affordable, and highly sensitive diagnostic tools. The central objective is to create an electrokinetic-driven lateral flow device that combines the simplicity of traditional rapid tests with advanced molecular sensing technologies. Utilizing electrically controlled fluid flow and highly specific DNA aptamers (synthetic receptors that bind malaria biomarkers with high precision), the device aims to deliver rapid, quantitative measurements of both parasite-derived and host-derived indicators of severe malaria. Such a tool would allow healthcare workers to diagnose malaria earlier, detect low-parasitemia infections that are often missed today, and assess the risk of severe disease directly at the point of care.

By integrating innovations in analytical chemistry, microfluidics, molecular biology, and clinical research, the project aims to provide a powerful solution adapted to both European and low-resource, malaria-endemic settings. The expected impact is significant: improving clinical decision-making, accelerating access to treatment, reducing healthcare costs linked to delayed diagnosis, and ultimately contributing to global efforts to reduce malaria morbidity and mortality. This approach aligns with international health priorities and supports the strategic goal of making advanced diagnostic technologies accessible to all.

Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti

During this reporting period, substantial progress was made in the development and characterization of DNA aptamers for the four malaria biomarkers central to MalDiProT. The work began with the fabrication and preliminary testing of a microfluidic SELEX chip; however, due to reproducibility issues, including clogging, incomplete fabrication of the amplification chamber, and contamination risks, the workflow was transitioned to a more reliable magnetic bead–based SELEX strategy. Using this optimized protocol, eight rounds of selection were performed independently for Ang-1 and Ang-2, incorporating extensive counter-selection steps (His-tag peptides, bare beads, and 10% serum) to eliminate non-specific binders. Next-generation sequencing identified highly enriched candidates, and biolayer interferometry confirmed strong, specific binding for several sequences. Two aptamers were ultimately selected as lead candidates: Ang-1.1 with picomolar affinity (KD = 11.7 pM) and no cross-reactivity, and Ang-2.13 with nanomolar affinity (KD = 3.76 nM) and similarly excellent specificity. These represent a major achievement, as no previously reported pairs have been shown to discriminate well between Ang-1 and Ang-2 without cross-reactivity.

In parallel, SELEX campaigns for PfHRP2 and PfLDH were completed using the same selection protocol, generating abundant candidate sequences. However, none exhibited detectable affinity for their targets. After repeating SELEX without improvement and considering project timelines, the contingency plan was implemented: previously published high-affinity aptamers for both biomarkers were retrieved and characterized. For PfHRP2, aptamer HRP2-702 produced a clear competitive LFA response spanning clinically relevant concentrations, with analytical parameters (LoD, LoQ, EC50, dynamic range) suitable for differentiating mild from severe malaria. For PfLDH, literature-reported aptamers P11-30 and P11-35 were validated by biolayer interferometry, confirming affinities of 18 and 39 nM, respectively. These aptamers are now ready for implementation in the electrokinetically driven LFA system.

Throughout the SELEX and validation stages, multiple technical challenges were addressed, including the optimization of PCR amplification steps, the adaptation of biolayer interferometry methods for screening large aptamer libraries, and the refinement of counter-selection strategies to ensure clinical specificity. In addition, I provided significant technical leadership within the host environment, training group members in AuNP conjugation, LFA strip dispenser operation, and aptamer-based assay fabrication. Overall, the project achieved its principal scientific goals for the outgoing phase: establishing a reproducible SELEX workflow, identifying two high-performance aptamers for Ang-1 and Ang-2, and preparing validated bioreceptors for PfHRP2 and PfLDH to be integrated into the next stages of assay development.

Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora)

The project has already delivered several advances that clearly move beyond the current state of the art in aptamer discovery and point-of-care diagnostics. First, the work led to the identification of two high-affinity DNA aptamers for Angiopoietin-1 (Ang-1) and Angiopoietin-2 (Ang-2), exhibiting picomolar and nanomolar affinities, respectively, with no observable cross-reactivity. This represents a significant scientific step forward, as all existing commercial and literature-reported binders for these clinically important inflammatory biomarkers exhibit unwanted cross-recognition, thereby preventing their independent quantification. The newly identified aptamers, therefore, open the door to accurate dual-biomarker sensing, enabling applications in malaria severity assessment and broader inflammatory diagnostics.

Additionally, the project showcased an innovative application of biolayer interferometry (BLI) as a rapid, quantitative method for validating aptamer affinity, specificity, and kinetic parameters. This provided the first complete kinetic profiles for these newly discovered aptamers, using association/dissociation modelling to extract high-quality KD, ka, and kdis values (e.g. Ang-1.1 KD ≈ 1.17 × 10⁻¹¹ M; Ang-2.13 KD ≈ 3.76 nM). Such kinetic resolution surpasses previous studies, which generally reported only endpoint binding or qualitative cross-reactivity tests. This positions the new aptamers as well-characterized bioreceptors ready for implementation in diagnostic platforms.

A second major contribution beyond the state of the art is the development of the first aptamer-based lateral flow assay (LFA) for vancomycin monitoring in dialysate samples from patients with acute kidney failure, performed during the outgoing phase with expertise transferred to the host lab. This represents a pioneering step in therapeutic drug monitoring at the point of care, presenting a low-cost alternative to the laboratory-based methods currently required for vancomycin quantification. This innovative diagnostic approach sets a precedent for expanding aptamer-based LFAs into therapeutic monitoring, a field still dominated by antibody-based laboratory assays.

The project also produced proof-of-concept advances in diagnostic biomarkers for malaria. Although SELEX for pfHRP2 and pfLDH did not yield novel aptamers, previously reported binders were rigorously re-characterised and integrated into prototype LFAs. For pfHRP2, a competitive AuNP-based LFA demonstrated a clear dose-response curve within clinically relevant concentration ranges, enabling the distinction between mild and severe malaria cases. For pfLDH, affinities of 18 and 39 nM were confirmed for aptamers P11-30 and P11-35, validating these receptors as suitable for high-sensitivity detection in future electrokinetic LFA formats. These results provide essential experimental evidence for integrating aptamers into low-resource malaria diagnostics.

Collectively, these advances place the project at the frontier of aptamer technology, microfluidics-inspired SELEX strategies, and next-generation point-of-care diagnostics. Key steps required for further uptake include microfluidic SELEX optimisation (to re-enable on-chip selection as originally planned), translation of aptamer-based LFAs to clinical samples, and industrial co-development to scale manufacturing of membranes, conjugates, and stable assay reagents. The project’s outputs are well-aligned with EU priorities in green, low-resource diagnostics, digital health, and reduced reliance on animal-derived antibodies, thereby strengthening European leadership in nucleic-acid-based diagnostic technologies.