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.

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.

NaNO3/NaCl Oxidant and Polyethylene Glycol (PEG) Capped Gold Nanoparticles (AuNPs) as a Novel Green Route for AuNPs Detection in Electrochemical Biosensors.

Gold nanoparticles (AuNPs) have been exploited as signal-producing tags in electrochemical biosensors. However, the electrochemical detection of AuNPs is currently performed using corrosive acid solutions, which may raise health and environmental concerns. Here, oxidant salts, and specifically the environmentally friendly and occupational safe NaNO3/NaCl mixture, have been evaluated for the first time as potential alternatives to the acid solutions traditionally used for AuNPs electrooxidation. In addition, a new strategy to improve the sensitivity of the biosensor through PEG-based ligand exchange to produce less compact and easier to oxidize AuNPs immunoconjugates is presented too. As we show, the electrochemical immunosensor using NaNO3/NaCl measurement solution for AuNPs electrooxidation and detection, coupled to the employment of PEG-capped nanoimmunoconjugates, produced results comparable to classical HCl detection. The procedure developed was next tested for human matrix metallopeptidase-9 (hMMP9) analysis, exhibiting a 0.18-23 ng/mL linear range, a detection limit of 0.06 ng/mL, and recoveries between 95 and 105% in spiked human plasma. These results show that the procedure developed is applicable to the analysis of protein biomarkers in blood plasma and could contribute to the development of more environmentally friendly AuNP-based electrochemical biosensors.

Electrochemical biosensing of non-electroactive targets using ferrocene-labeled magnetic particles and CNT wiring.

We show that target binding onto ferrocene-modified magnetic microparticles (MP-Fc) promotes physical sheltering of the labels. This can be measured electrochemically by CNT wiring, which enhances ten-fold the signals registered compared to direct detection of the MPs alone. As a proof of concept, detection of detergents and antibodies is accomplished. In these preliminary experiments, random binding of 0.01% Tween 20 onto MP-Fc was detectable both voltametrically and impedimetrically after a 2 min incubation. Furthermore, affinity capture of 4 µg mL(-1) of biotinylated antibody by streptavidin MP-Fc could be measured in less than 30 min and even in the presence of 1 mg mL(-1) of BSA.

Carbon nanotube wiring for signal amplification of electrochemical magneto immunosensors: application to myeloperoxidase detection.

In this work, chronoamperometric myelo-peroxidase (MPO) detection was accomplished using immunofunctionalized magnetic microparticles (MPs), disposable carbon screen-printed electrodes (C-SPEs), and a ready-to-use commercially available tetramethylbenzidine (TMB)-based enzymatic substrate. In order to reach the limit of detection (LOD) needed to study real blood serum samples, assay performance was additionally improved by exploiting CNT wiring, which amplified the signal and decreased the LOD. The optimized assay can be performed in 30 min and yields LODs of 6 and 55 ng mL(-1) in PBS and undiluted human serum, respectively, making it useful for the identification of patients at risk of cardiovascular disease. These results demonstrate that electrode nanostructuring can be accomplished "post-assay," which favors the development of enhanced magneto immunosensors based on the exploitation of cheap and simple SPE devices.

Effect of early initiation of noninvasive ventilation in patients transported by emergency medical service for acute heart failure.

BACKGROUND: While the indication for noninvasive ventilation (NIV) in severely hypoxemic patients with acute heart failure (AHF) is often indicated and may improve clinical course, the benefit of early initiation before patient arrival to the emergency department (ED) remains unknown. OBJECTIVE: This study aimed to assess the impact of early initiation of NIV during emergency medical service (EMS) transportation on outcomes in patients with AHF. DESIGN: A secondary retrospective analysis of the EAHFE (Epidemiology of AHF in EDs) registry. SETTING: Fifty-three Spanish EDs. PARTICIPANTS: Patients with AHF transported by EMS physician-staffed ambulances who were treated with NIV at any time during of their emergency care were included and categorized into two groups based on the place of NIV initiation: prehospital (EMS group) or ED (ED group). OUTCOME MEASURES: Primary outcome was the composite of in-hospital mortality and 30-day postdischarge death, readmission to hospital or return visit to the ED due to AHF. Secondary outcomes included 30-day all-cause mortality after the index event (ED admission) and the different component of the composite primary endpoint considered individually. Multivariate logistic regressions were employed for analysis. RESULTS: Out of 2406 patients transported by EMS, 487 received NIV (EMS group: 31%; EMS group: 69%). Mean age was 79 years, 48% were women. The EMS group, characterized by younger age, more coronary artery disease, and less atrial fibrillation, received more prehospital treatments. The adjusted odds ratio (aOR) for composite endpoint was 0.66 (95% CI: 0.42-1.05). The aOR for secondary endpoints were 0.74 (95% CI: 0.38-1.45) for in-hospital mortality, 0.74 (95% CI: 0.40-1.37) for 30-day mortality, 0.70 (95% CI: 0.41-1.21) for 30-day postdischarge ED reconsultation, 0.80 (95% CI: 0.44-1.44) for 30-day postdischarge rehospitalization, and 0.72 (95% CI: 0.25-2.04) for 30-day postdischarge death. CONCLUSION: In this ancillary analysis, prehospital initiation of NIV in patients with AHF was not associated with a significant reduction in short-term outcomes. The large confidence intervals, however, may preclude significant conclusion, and all point estimates consistently pointed toward a potential benefit from early NIV initiation.

Chronoamperometric magneto immunosensor for myeloperoxidase detection in human plasma based on a magnetic switch produced by 3D laser sintering.

In this work, an amperometric immunosensor for detection of myeloperoxidase (MPO) in human plasma is reported. Detection is based on the immobilization of anti-MPO antibodies onto magnetic beads (MBs). Following MPO immunocapture and washing steps, MBs are transferred to a customized modular detector device produced by 3D laser sintering. This tool integrates electrodes, electrical connectors, and a novel magnetic switch, whose functioning is founded on the vertical displacement of a permanent magnet. In this way, magnetic switching makes possible the confinement of MBs over the working electrode for electrochemical detection, followed by the release of MBs for electrode washing and reutilization. Notably, electrochemical detection is based on the endogenous MPO activity, which reduces reagent consumption and assay time compared to sandwich assays using enzyme-labeled antibodies. After optimization, the assay could be completed in 45 min and displayed a linear response between 0.9 and 60 ng mL(-1) for MPO and a limit of detection of 0.4 ng mL(-1). The real applicability of this approach is demonstrated by the ability to carry out the successful analysis of MPO in human plasma samples. Furthermore, the results allowed the classification of patients into three groups at risk of suffering cardiac events (i.e., low, medium, or high) and correlated well with data provided by a commercially available standardized method.

Fast immunosensing technique to detect Legionella pneumophila in different natural and anthropogenic environments: comparative and collaborative trials.

BACKGROUND: Legionellosis is an uncommon form of pneumonia. After a clinical encounter, the necessary antibiotic treatment is available if the diagnosis is made early in the illness. Before the clinical encounter, early detection of the main pathogen involved, Legionella pneumophila, in hazardous environments is important in preventing infectious levels of this bacterium. In this study a qualitative test based on combined magnetic immunocapture and enzyme-immunoassay for the fast detection of Legionella pneumophila in water samples was compared with the standard method, in both comparative and collaborative trials. The test was based on the use of anti-Legionella pneumophila antibodies immobilized on magnetic microspheres. The final protocol included concentration by filtration, resuspension and immunomagnetic capture. The whole assay took less than 1 hour to complete. RESULTS: A comparative trial was performed against the standard culture method (ISO 11731) on both artificially and naturally contaminated water samples, for two matrices: chlorinated tap water and cooling tower water. Performance characteristics of the test used as screening with culture confirmation resulted in sensitivity, specificity, false positive, false negative, and efficiency of 96.6%, 100%, 0%, 3.4%, and 97.8%, respectively. The detection limit at the level under which the false negative rate increases to 50% (LOD50) was 93 colony forming units (CFU) in the volume examined for both tested matrices. The collaborative trial included twelve laboratories. Water samples spiked with certified reference materials were tested. In this study the coincidence level between the two methods was 95.8%. CONCLUSION: Results demonstrate the applicability of this immunosensing technique to the rapid, simple, and efficient detection of Legionella pneumophila in water samples. This test is not based on microbial growth, so it could be used as a rapid screening technique for the detection of L. pneumophila in waters, maintaining the performance of conventional culture for isolation of the pathogen and related studies.

Voltammetric discrimination of skatole and indole at disposable screen printed electrodes.

In this work, we study the electrochemical behaviour of skatole, one of the compounds responsible for the offensive smell in pork meat that is known as boar taint, at different metal and carbon electrodes. We then demonstrate for the first time that skatole and indole, the main electroactive interferent potentially present in real samples, can be discriminated and separately quantified using cheap and disposable screen printed electrodes (SPE). This implies significant progress compared to the colorimetric method reported by Mortensen in 1983.

Magnetic Bead Handling Using a Paper-Based Device for Quantitative Point-of-Care Testing.

Microfluidic paper-based analytical devices (µPADs) have been extensively proposed as ideal tools for point-of-care (POC) testing with minimal user training and technical requirements. However, most µPADs use dried bioreagents, which complicate production, reduce device reproducibility and stability, and require transport and storage under temperature and humidity-controlled conditions. In this work, we propose a µPAD produced using an affordable craft-cutter and stored at room temperature, which is used to partially automate a single-step colorimetric magneto-immunoassay. As a proof-of-concept, the µPAD has been applied to the quantitative detection of Plasmodium falciparum lactate dehydrogenase (Pf-LDH), a biomarker of malaria infection. In this system, detection is based on a single-step magneto-immunoassay that consists of a single 5-min incubation of the lysed blood sample with immuno-modified magnetic beads (MB), detection antibody, and an enzymatic signal amplifier (Poly-HRP). This mixture is then transferred to a single-piece paper device where, after on-chip MB magnetic concentration and washing, signal generation is achieved by adding a chromogenic enzyme substrate. The colorimetric readout is achieved by the naked eye or using a smartphone camera and free software for image analysis. This µPAD afforded quantitative Pf-LDH detection in <15 min, with a detection limit of 6.25 ng mL−1 when the result was interpreted by the naked eye and 1.4 ng mL−1 when analysed using the smartphone imaging system. Moreover, the study of a battery of clinical samples revealed concentrations of Pf-LDH that correlated with those provided by the reference ELISA and with better sensitivity than a commercial rapid diagnostic test (RDT). These results demonstrate that magneto-immunoassays can be partly automated by employing a µPAD, achieving a level of handling that approaches the requirements of POC testing.

Radiography-based triage for COVID-19 in the Emergency Department in a Spanish cohort of patients.

Background: Strategies to determine who could be safely discharged home from the Emergency Department (ED) in COVID-19 are needed to decongestion healthcare systems. Objectives: To describe the outcomes of an ED triage system for non-severe patients with suspected COVID-19 and possible pneumonia based on chest X-ray (CXR) upon admission. Material and methods: Retrospective, single-center study performed in Barcelona (Spain) during the COVID-19 peak in March-April 2020. Patients with COVID-19 symptoms and potential pneumonia, without respiratory insufficiency, with priority class IV-V (Andorran triage model) had a CXR upon admission. This approach tried to optimize resource use and to facilitate discharges. The results after adopting this organizational approach are reported. Results: We included 834 patients, 53% were female. Most patients were white (66%) or Hispanic (27%). CXR showed pneumonia in 523 (62.7%). Compared to those without pneumonia, patients with pneumonia were older (55 vs 46.6 years old) and had a higher Charlson comorbidity index (1.9 vs 1.3). Patients with pneumonia were at a higher risk for a combined outcome of admission and/or death (91 vs 12%). Death rates tended to be numerically higher in the pneumonia group (10 vs 1). Among patients without pneumonia in the initial CXR, 10% reconsulted (40% of them with new pneumonia). Conclusion: CXR identified pneumonia in a significant number of patients. Those without pneumonia were mostly discharged. Mortality among patients with an initially negative CXR was low. CXR triage for pneumonia in non-severe COVID-19 patients in the ED can be an effective strategy to optimize resource use.

Introducción: La pandemia de COVID-19 conlleva una alta ocupación de los servicios de urgencias (SU). Se requieren nuevas estrategias para optimizar la gestión de estos recursos. Objetivos: Describir los resultados de un sistema de triaje en urgencias para pacientes no graves con sospecha de COVID-19 y posible neumonía, basado en la radiografía de tórax (RT). Material y métodos: Estudio retrospectivo, unicéntrico realizado en Barcelona (España) entre marzo y abril de 2020. Se realizó una RT al ingreso en SU de pacientes con síntomas de COVID-19 y sospecha de neumonía, sin insuficiencia respiratoria, con una prioridad clase IV-V (sistema andorrano de triaje). Esta medida pretende optimizar los recursos y facilitar las altas. Se reportan los resultados tras adoptar esta estrategia. Resultados: Se incluyeron 834 pacientes (53% mujeres, 66% caucásicos, 27% hispánicos). La RT mostró neumonía en 523 (62,7%). Comparados con los pacientes sin neumonía, los que sí la padecían eran mayores (55 vs. 46,6 años), con un índice de comorbilidad de Charlson más elevado (1,9 vs. 1,3) y con mayor riesgo de ingreso y/o muerte (91 vs. 12%). La mortalidad fue numéricamente mayor en el grupo con neumonía (10 vs. 1). El 10% de los pacientes sin neumonía en RT consultaron de nuevo al SU (40% con neumonía). Conclusión: La RT identificó neumonía en múltiples pacientes. Los que no tenían neumonía fueron mayoritariamente dados de alta. La mortalidad entre pacientes con RT negativa fue baja. La RT como triaje para neumonía en pacientes con COVID-19 no grave puede ahorrar recursos.

Radiography-based triage for COVID-19 in the Emergency Department in a Spanish cohort of patients.

BACKGROUND: Strategies to determine who could be safely discharged home from the Emergency Department (ED) in COVID-19 are needed to decongestion healthcare systems. OBJECTIVES: To describe the outcomes of an ED triage system for non-severe patients with suspected COVID-19 and possible pneumonia based on chest X-ray (CXR) upon admission. MATERIAL AND METHODS: Retrospective, single-center study performed in Barcelona (Spain) during the COVID-19 peak in March-April 2020. Patients with COVID-19 symptoms and potential pneumonia, without respiratory insufficiency, with priority class IV-V (Andorran triage model) had a CXR upon admission. This approach tried to optimize resource use and to facilitate discharges. The results after adopting this organizational approach are reported. RESULTS: We included 834 patients, 53% were female. Most patients were white (66%) or Hispanic (27%). CXR showed pneumonia in 523 (62.7%). Compared to those without pneumonia, patients with pneumonia were older (55 vs 46.6 years old) and had a higher Charlson comorbidity index (1.9 vs 1.3). Patients with pneumonia were at a higher risk for a combined outcome of admission and/or death (91 vs 12%). Death rates tended to be numerically higher in the pneumonia group (10 vs 1). Among patients without pneumonia in the initial CXR, 10% reconsulted (40% of them with new pneumonia). CONCLUSION: CXR identified pneumonia in a significant number of patients. Those without pneumonia were mostly discharged. Mortality among patients with an initially negative CXR was low. CXR triage for pneumonia in non-severe COVID-19 patients in the ED can be an effective strategy to optimize resource use.

Carbon nanotube wiring: a tool for straightforward electrochemical biosensing at magnetic particles.

Here, we combine the unique properties of carbon nanotubes (CNTs) and magnetic particles (MPs) to develop a novel biosensing approach for the specific detection of electroactive labels and targets. The assay is based on label/target capture and concentration using MPs. It follows addition of CNTs, which adsorb onto the surface of the beads. The subsequent magnetic entrapment of the CNT/MP complexes onto an electrode allows straightforward electrochemical sensing of the MP surface by exploiting CNT wiring. As a proof of concept, the assay has been applied to detection of ferrocene labels, and to the specific immunodetection of dopamine in both artificial saline solutions and real sample matrixes. The results demonstrate the applicability of CNT as wiring tools for enzymeless and substrateless electrochemical biosensing.

Electrochemical detection of quorum sensing signaling molecules by dual signal confirmation at microelectrode arrays.

n-Acyl homoserine lactones (AHLs) are produced by gram-negative bacteria to regulate gene expression in a cell density dependent manner. For instance, expression of virulence factors by pathogens such as Pseudomonas aeruginosa is induced only when a threshold concentration of AHLs is reached, which indicates that the bacterial population is big enough to promote infection. In this study, the indicator strain Agrobacterium tumefaciens NTL4 (pZLR4), which carries a ß-galactosidase (ß-gal) reporter gene under the control of a quorum sensing promoter, was used to develop an electrochemical biosensor to detect AHLs using the model n-(3-oxo)-dodecanoyl-L-homoserine lactone (oxo-C12-HSL), an AHL previously detected in cystic fibrosis patients infected with P. aeruginosa. The substrate 4-aminophenyl ß-D-galactopyranoside was used to detect ß-gal activity by cyclic voltammetry. Furthermore, simultaneous monitoring of substrate consumption and p-aminophenol production by ß-gal allowed on-chip result verification by dual-signal confirmation. The sensor exhibited high reproducibility and accurately detected oxo-C12-HSL in a low picomolar to low nanomolar range in spiked liquid cultures and artificial saliva, as well as AHLs naturally released by P. aeruginosa in culture supernatants. Moreover, detection took just 2 h, required no sample pretreatment or preconcentration steps, and was easier and faster than traditional methods.

Electrochemical detection of testosterone by use of three-dimensional disc-ring microelectrode sensing platforms: application to doping monitoring.

Testosterone is one of the androgenic steroid hormones, the consumption of which is considered doping in most sports. Here, we present powerful 3D sensing platforms using novel disc-ring microelectrode array devices and exploit them for the competitive immunosensing of testosterone. Each device contains a microelectrode array that consists of a large number of individual microdiscs and is used as the substrate for immunofunctionalization and assay performance. One micrometer above it, a second microelectrode array, this time consisting of microrings, is used as the working electrode for electrochemical monitoring. The physical separation of these two functions allows the incorporation of relatively thick biocomponent layers during immunofunctionalization of the microdiscs without negatively affecting electrochemical detection at the rings. Moreover, it permits electrochemical activation of the latter immediately before substrate addition and hence enables optimal electrode performance. The optimized assay showed a linear range between 0.01 and 10 ng/mL and a limit of detection of 12.5 pg/mL testosterone with detection times of 45 min.

Smartphone-Enabled Personalized Diagnostics: Current Status and Future Prospects.

Smartphones are becoming increasingly versatile thanks to the wide variety of sensor and actuator systems packed in them. Mobile devices today go well beyond their original purpose as communication devices, and this enables important new applications, ranging from augmented reality to the Internet of Things. Personalized diagnostics is one of the areas where mobile devices can have the greatest impact. Hitherto, the camera and communication abilities of these devices have been barely exploited for point of care (POC) purposes. This short review covers the recent evolution of mobile devices in the area of POC diagnostics and puts forward some ideas that may facilitate the development of more advanced applications and devices in the area of personalized diagnostics. With this purpose, the potential exploitation of wireless power and actuation of sensors and biosensors using near field communication (NFC), the use of the screen as a light source for actuation and spectroscopic analysis, using the haptic module to enhance mass transport in micro volumes, and the use of magnetic sensors are discussed.

Detection of Viruses and Virus-Neutralizing Antibodies Using Synthetic Erythrocytes: Toward a Tuneable Tool for Virus Surveillance.

The hemagglutination inhibition assay (HAI) is a classical method used worldwide in many analytical applications, including pathogen identification, vaccine production monitoring, and detection and characterization of pathogen-neutralizing antibodies (n-Ab). This is also a World Health Organization (WHO) reference method for the global surveillance of influenza viruses, which provides the information needed for the annual reformulation of the flu vaccine. HAI is a simple and inexpensive method that is performed without sophisticated equipment. However, it has to be carried out with fresh red blood cells (RBCs), a highly variable, unstable, and hard to mass-produce reagent, which impairs assay reproducibility. Here, we used the tests employed for influenza surveillance as a model to develop synthrocytes©, a synthetic reagent that could substitute animal erythrocytes in HAI. Contrary to previous examples exploiting sophisticated production paths to generate therapeutic synthetic RBCs, we founded production on the identification of microparticles able to generate different sedimentation patterns when agglutinated or not, which is the main requirement for HAI testing. Upon incorporation of influenza-binding receptors and optimization of production and assay conditions, synthrocytes succeeded in binding influenza A(H1N1) and B viruses as erythrocytes do, but were faster and more stable. Synthrocytes were finally employed in an HAI-like assay to detect the WHO reference reagents for influenza surveillance. Our results show that it is possible to substitute erythrocytes in classical HAI by a highly tuneable and potentially mass-produced synthetic reagent, which should facilitate worldwide HAI standardization with minimal equipment or training requirements.

Electrochemical Capillary-Flow Immunoassay for Detecting Anti-SARS-CoV-2 Nucleocapsid Protein Antibodies at the Point of Care.

Rapid and inexpensive serological tests for SARS-CoV-2 antibodies are needed to conduct population-level seroprevalence surveillance studies and can improve diagnostic reliability when used in combination with viral tests. Here, we report a novel low-cost electrochemical capillary-flow device to quantify IgG antibodies targeting SARS-CoV-2 nucleocapsid proteins (anti-N antibody) down to 5 ng/mL in low-volume (10 µL) human whole blood samples in under 20 min. No sample preparation is needed as the device integrates a blood-filtration membrane for on-board plasma extraction. The device is made of stacked layers of a hydrophilic polyester and double-sided adhesive films, which create a passive microfluidic circuit that automates the steps of an enzyme-linked immunosorbent assay (ELISA). The sample and reagents are sequentially delivered to a nitrocellulose membrane that is modified with a recombinant SARS-CoV-2 nucleocapsid protein. When present in the sample, anti-N antibodies are captured on the nitrocellulose membrane and detected via chronoamperometry performed on a screen-printed carbon electrode. As a result of this quantitative electrochemical readout, no result interpretation is required, making the device ideal for point-of-care (POC) use by non-trained users. Moreover, we show that the device can be coupled to a near-field communication potentiostat operated from a smartphone, confirming its true POC potential. The novelty of this work resides in the integration of sensitive electrochemical detection with capillary-flow immunoassay, providing accuracy at the point of care. This novel electrochemical capillary-flow device has the potential to aid the diagnosis of infectious diseases at the point of care.

Detection of Plasmodium falciparum malaria in 1 h using a simplified enzyme-linked immunosorbent assay.

Malaria is a parasitic disease caused by protists of the genus Plasmodium, which are transmitted to humans through the bite of infected female Anopheles mosquitoes. Analytical methodologies and efficient drugs exist for the early detection and treatment of malaria, and yet this disease continues infecting millions of people and claiming several hundred thousand lives each year. One of the reasons behind this failure to control the disease is that the standard method for malaria diagnosis, microscopy, is time-consuming and requires trained personnel. Alternatively, rapid diagnostic tests, which have become common for point-of-care testing thanks to their simplicity of use, tend to be insufficiently sensitive and reliable, and PCR, which is sensitive, is too complex and expensive for massive population screening. In this work, we report a sensitive simplified ELISA for the quantitation of Plasmodium falciparum lactate dehydrogenase (Pf-LDH), which is capable of detecting malaria in 45-60 min. Assay development was founded in the selection of high-performance antibodies, implementation of a poly-horseradish peroxidase (polyHRP) signal amplifier, and optimization of whole-blood sample pre-treatment. The simplified ELISA achieved limits of detection (LOD) and quantification (LOQ) of 0.11 ng mL-1 and 0.37 ng mL-1, respectively, in lysed whole blood, and an LOD comparable to that of PCR in Plasmodium in vitro cultures (0.67 and 1.33 parasites µL-1 for ELISA and PCR, respectively). Accordingly, the developed immunoassay represents a simple and effective diagnostic tool for P. falciparum malaria, with a time-to-result of <60 min and sensitivity similar to the reference PCR, but easier to implement in low-resource settings.

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.