Development of a Fast Chemiluminescent Magneto-Immunoassay for Sensitive Plasmodium falciparum Detection in Whole Blood.

The World Health Organization (WHO) estimates that over three billion people are at risk of acquiring malaria, a parasitic infection that produces more than 200 million new infections and nearly half a million deaths each year. Expanding the access to early diagnosis and treatment is one of the most effective ways to prevent disease complications, reduce patient mortality, and curb the community transmission. However, none of the diagnostic methods used currently for malaria detection, including light microscopy, polymerase chain reaction (PCR), and rapid diagnostic tests (RDTs), can provide simultaneously fast results, high sensitivity, and parasitaemia quantitation with minimal user intervention. Here, we present a magneto-immunoassay that, based on the unique combination of magnetic beads (MB), an enzymatic signal amplifier (Poly-HRP), and chemiluminescence detection, provides fast, sensitive, and quantitative malaria diagnosis with easy user manipulation. This assay quantifies Plasmodium falciparum lactate dehydrogenase (PfLDH) in lysed whole blood samples in <15 min, exhibiting a limit of detection (LOD) of 0.02 ng mL-1 and providing patient stratification consistent with the reference methods. These figures of merit surpass the performance of the magneto-immunoassays reported previously for Plasmodium detection and demonstrate for the first time that the proposed combination of MB, Poly-HRP, and chemiluminescence detection produces extremely fast, simple, and efficient assays that approach the requirements of point-of-care (POC) malaria surveillance.

Electrochemical POC device for fast malaria quantitative diagnosis in whole blood by using magnetic beads, Poly-HRP and microfluidic paper electrodes.

Malaria, a parasitic infection caused by Plasmodium parasites and transmitted through the bite of infected female Anopheles mosquitos, is one of the main causes of mortality in many developing countries. Over 200 million new infections and nearly half a million deaths are reported each year, and more than three billion people are at risk of acquiring malaria worldwide. Nevertheless, most malaria cases could be cured if detected early. Malaria eradication is a top priority of the World Health Organisation. However, achieving this goal will require mass population screening and treatment, which will be hard to accomplish with current diagnostic tools. We report an electrochemical point-of-care device for the fast, simple and quantitative detection of Plasmodiumfalciparum lactate dehydrogenase (PfLDH) in whole blood samples. Sample analysis includes 5-min lysis to release intracellular parasites, and stirring for 5 more min with immuno-modified magnetic beads (MB) along with an immuno-modified signal amplifier. The rest of the magneto-immunoassay, including sample filtration, MB washing and electrochemical detection, is performed at a disposable paper electrode microfluidic device. The sensor provides PfLDH quantitation down to 2.47 ng mL-1 in spiked samples and for 0.006-1.5% parasitemias in Plasmodium-infected cultured red blood cells, and discrimination between healthy individuals and malaria patients presenting parasitemias >0.3%. Quantitative malaria diagnosis is attained with little user intervention, which is not achieved by other diagnostic methods.

Competitive USB-Powered Hand-Held Potentiostat for POC Applications: An HRP Detection Case.

Considerable efforts are made to develop Point-of-Care (POC) diagnostic tests. POC devices have the potential to match or surpass conventional systems regarding time, accuracy, and cost, and they are significantly easier to operate by or close to the patient. This strongly depends on the availability of miniaturized measurement equipment able to provide a fast and sensitive response. This paper presents a low-cost, portable, miniaturized USB-powered potentiostat for electrochemical analysis, which has been designed, fabricated, characterized, and tested against three forms of high-cost commercial equipment. The portable platform has a final size of 10.5 × 5.8 × 2.5 cm, a weight of 41 g, and an approximate manufacturing cost of $85 USD. It includes three main components: the power module which generates a stable voltage and a negative supply, the front-end module that comprises a dual-supply potentiostat, and the back-end module, composed of a microcontroller unit and a LabVIEW-based graphic user interface, granting plug-and-play and easy-to-use operation on any computer. The performance of this prototype was evaluated by detecting chronoamperometrically horseradish peroxidase (HRP), the enzymatic label most widely used in electrochemical biosensors. As will be shown, the miniaturized platform detected HRP at concentrations ranging from 0.01 ng·mL-1 to 1 µg·mL-1, with results comparable to those obtained with the three commercial electrochemical systems.

Detection of plasma MMP-9 within minutes. Unveiling some of the clues to develop fast and simple electrochemical magneto-immunosensors.

Magnetic beads (MB) have been extensively used to produce sensitive and efficient electrochemical magneto-immunosensors. However, MB effective handling requires training, and MB washing after each incubation step is time consuming and contributes to raise result variability. Consequently, most of the electrochemical magneto-immunosensors reported to date, which entailed relatively long and complex multi-step procedures, would be difficult to carry out at point-of-care (POC) settings or by laypersons. For this reason, here we targeted the development of a simplified detection path, which is fast and simple enough to be operated at a POC setting, sufficiently efficient to provide analyte quantitation comparable to classical diagnostic methods, and dependent on minimal technical requirements to facilitate method global exploitation. As a proof-of-concept, we optimized an extremely simple, fast and efficient electrochemical magneto-immunosensor for detection of matrix metalloproteinase 9 (MMP-9). To accomplish this, we optimized MB immunomodification, produced an immunomodified Poly-HRP signal amplifier, developed a single-step magneto-immunoassay, and optimized electrochemical detection using a multiplexed magnetic holder and a ready-to-use commercial substrate solution. The sensor was finally calibrated by detecting MMP-9 in clinical samples. This electrochemical magneto-immunosensor detected MMP-9 in just 12-15 min, displaying linear response between 0.03 and 2 ng mL-1 of MMP-9, limits of detection (LOD) and quantification (LOQ) of 13 pg mL-1 and 70 pg mL-1, respectively, %CV< 6%, and accurate quantification of MMP-9 in patient plasma samples. These results were comparable to those afforded by a 5-h reference ELISA that used the same antibodies, confirming the applicability of our simplified method.

Using magnetic beads and signal amplifiers to produce short and simple immunoassays: Application to MMP-9 detection in plasma samples.

Magnetic beads (MB) and signal amplifiers, such as horseradish peroxidase polymers (poly-HRP), have been used before for the production of highly sensitive immunoassays. However, most of the examples reported previously entailed long and tedious multi-step procedures, which were not necessarily shorter or simpler than classical paths such as Enzyme-Linked Immunosorbent Assay (ELISA). Here, instead of exploiting the combination of MB and poly-HRP to ameliorate sensitivity, we show that they conform a powerful tool that can be used to shorten the incubation times, which allows optimizing extremely simple, fast and efficient immunoassays with minimal technical requirements. In order to do so, here we used the highly sensitive and specific pair of antibodies of a commercial ELISA kit to optimize a magneto-ELISA for the detection of matrix metallopeptidase 9 (MMP-9). Three signal amplifiers were then tested and the best performing one was implemented in the magneto-assay to shorten the incubation times and improve assay performance. As we show, the shortened magneto-assay could be carried out in about 35 min, which included two 5-min incubations, washing, and incubation with enzyme substrate for 20 min before colorimetric detection. Moreover, the quantification of MMP-9 provided by the shortened assay in 12 plasma samples collected from patients was comparable to that generated by the 5-h ELISA, which was 8.5 times longer.