Paper-based microfluidics for DNA diagnostics of malaria in low resource underserved rural communities

Abstract

Rapid, low-cost, species-specific diagnosis, based upon DNA testing, is becoming important in the treatment of patients with infectious diseases. Here, we demonstrate an innovation that uses origami to enable multiplexed, sensitive assays that rival polymerase chain reactions (PCR) laboratory assays and provide high-quality, fast precision diagnostics for malaria. The paper-based microfluidic technology proposed here combines vertical flow sample-processing steps, including paper folding for whole-blood sample preparation, with an isothermal amplification and a lateral flow detection, incorporating a simple visualization system. Studies were performed in village schools in Uganda with individual diagnoses being completed in <50 min (faster than the standard laboratory-based PCR). The tests, which enabled the diagnosis of malaria species in patients from a finger prick of whole blood, were both highly sensitive and specific, detecting malaria in 98% of infected individuals in a double-blind first-in-human study. Our method was more sensitive than other field-based, benchmark techniques, including optical microscopy and industry standard rapid immunodiagnostic tests, both performed by experienced local healthcare teams (which detected malaria in 86% and 83% of cases, respectively). All assays were independently validated using a real-time double-blinded reference PCR assay. We not only demonstrate that advanced, low-cost DNA-based sensors can be implemented in underserved communities at the point of need but also highlight the challenges associated with developing and implementing new diagnostic technologies in the field, without access to laboratories or infrastructure.

Lab-on-a-Chip Technologies for the Single Cell Level: Separation, Analysis, and Diagnostics

“…Some salient examples thereof are circulating tumor cells (CTC) of various types of cancer [1,[36][37][38], rare cells (e.g., sickle-cell variants of red blood cells) [39,40], parasites, like Plasmodium falciparum [1,7,10,11,[13][14][15][21][22][23]36,[41][42][43][44][45][46][47][48][49][50] and Trypanosoma spp. [8,44,[51][52][53][54][55][56][57] and even plant pathogens [26,58], as well as-after cells have been lysed-subcellular infection markers (e.g., DNA, RNA fragments) [10,11,22,43,[59][60][61][62]. Given the vast adaptability of microfluidics to any kind of single or multi-cellular assay [63], the ability to combine it with various light microscopy techniques…”

“…Since paper is, on the microscopic scale, a tangle of fibers, most cells and similarly big particles are retained, while the smaller and soluble parts travel along the paper as they do in thin layer chromatography (see Figure 10). Thus, PMF is used more often to analyze the contents of lysed cells (e.g., DNA [11,16,20]) than entire cells. However, it also possible to have a PMF device that starts out with a sample of cells and media, first separates the cells, then lyses the isolated cells and runs molecular analysis on them.…”

“…However, it also possible to have a PMF device that starts out with a sample of cells and media, first separates the cells, then lyses the isolated cells and runs molecular analysis on them. One example is a field-applicable test using foldable paper slips for diagnosis of Malaria in infected blood samples [11].…”

“…One very prominent aspect of microfluidic research is diagnostics on the single-cell or even molecular level. The significance of recent research towards microfluidics-based single cell diagnostic chips is apparent in health care, in our homes, and also very prominently in the fight against the COVID 19 pandemic: The diagnostic targets range from circulating tumor cells (CTC) [1][2][3][4][5][6], over parasites in blood [7][8][9][10][11][12][13][14][15], male fertility [16][17][18][19][20], molecular markers for infections [11,15,[21][22][23], cells of a specific stage in their life cycle [24,25], plant pathogens [26] and the SARS-CoV-2 proteome [27][28][29][30][31][32]. Depending on the exact target (either the entire cell or sub-cellular markers) there are different approaches to on-chip detection, each with their own underlying fundamentals and set of limitations.…”

Ultra-Low-Cost Integrated Silicon-based Transducer for On-Site, Genetic Detection of Pathogens

“…Optical detection systems have been difficult to miniaturize and largely limited to centralized laboratories, however, some early concepts have been proposed by various groups in the literature. [5][6][7][8][9][10] Despite low-cost onsite testing for infectious diseases is being the holy grail of NA diagnostics, there are still no inexpensive and handheld solutions in the market that can provide truly portable, rapid NA detection. Commercially available, benchtop optical lab instruments such as GeneXpert (Cephied) have already made a substantial difference in the speed of NA-based diagnosis of infectious diseases but the high cost of the instrument/tests, large size and power consumption have prevented the adoption of these systems for use in the field.…”