Handheld Purification-Free Nucleic Acid Testing Device for Point-of-Need Detection of Malaria from Whole Blood

Abstract

World Health Organization’s aim to eliminate malaria from developing/resource-limited economies requires easy access to low-cost, highly sensitive, and specific screening. We present a handheld nucleic acid testing device with on-chip automated sample preparation to detect malaria (Plasmodium falciparum) infection from a whole blood sample as a feasibility study. We used a simple two-reagent-based purification-free protocol to prepare the whole blood sample on a piezo pump pressure-driven microfluidic cartridge. The cartridge includes a unique mixing chamber for sample preparation and metering structures to dispense a predetermined volume of the sample lysate mixture into four chambers containing a reaction mix. The parasite genomic DNA concentration can be estimated by monitoring the fluorescence generated from the loop-mediated isothermal amplification reaction in real time. We achieved a sensitivity of ∼0.42 parasite/μL of whole blood, sufficient for detecting asymptomatic malaria parasite carriers.

Intelligent point of care testing for medicine diagnosis

“…In 2023, Kshirsagar et al presented a novel handheld nucleic acid detection device with integrated onchip sample preparation for the detection of Plasmodium falciparum, the causative agent of severe malaria, from whole blood samples (Figure 3C). 33 This technology employs a simple purification-free approach, utilizing two reagents for the preparation of whole blood samples on a microfluidic cartridge driven by a piezoelectric pump. By utilizing this device, accurate detection of asymptomatic malaria carriers is enabled.…”

“…Reproduced with permission. 33 Copyright 2023, American Chemical Society. (D) The design of the capillary-based microfluidic chip.…”

“…(C) Schematic diagram of the structure of a handheld purification free nucleic acid detector. Reproduced with permission 33 . Copyright 2023, American Chemical Society.…”

Dual CRISPR/Cas13a Cascade Strand Displacement-Triggered Transcription for Point-of-Care Detection of Plasmodium in Asymptomatic Malaria

“…Polymerase chain reaction (PCR)-based methods are currently considered as the gold standard for the detection of asymptomatic malaria in centralized laboratories. However, the requirement of thermal cycling and sophisticated equipment restricts their applicability in resource-constrained regions. , Isothermal nucleic acid amplification techniques, such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP), have emerged as user-friendly substitutes for PCR. , The isothermal reaction process of these amplification methods simplifies equipment requirements, making them suitable for deployment in resource-limited areas. , Nonetheless, false positive signals in isothermal amplification methods resulting from nonspecific amplification hinder their widespread adoption. , Therefore, accurate POC diagnostics are also eagerly required.…”

“…17,18 The isothermal reaction process of these amplification methods simplifies equipment requirements, making them suitable for deployment in resource-limited areas. 19,20 Nonetheless, false positive signals in isothermal amplification methods resulting from nonspecific amplification hinder their widespread adoption. 21,22 Therefore, accurate POC diagnostics are also eagerly required.…”