Malaria Biomarker Research Articles

Microfluidic device: A versatile biosensor platform to multiplex aptamer-based detection of malaria biomarkers.

Plasmodium falciparum malaria remains a dominant infectious disease that affects Africa than the rest of the world, considering its associated cases and death rates. It's a febrile illness that produces several reliable biomarkers, for example, P. falciparum lactate dehydrogenase (PfLDH), P. falciparum Plasmodium glutamate dehydrogenase (PfGDH), and P. falciparum histidine-rich proteins (HRP-II) in blood circulatory system that can easily be employed as targets in rapid diagnostic tests (RDTs). In recent times, several DNA aptamers have been developed via SELEX technology to detect some specific malaria biomarkers (PfLDH, PvLDH, HRP-II, PfGDH) in a biosensor mode with good binding affinity properties to overcome the trend of cross-reactivity, limited sensitivity and stability problems that have been observed with immunodiagnostics. In this review, we summarized existing diagnostic methods and relevant biomarkers to suggest promising approaches to develop sensitive and species-specific multiplexed diagnostic devices enabling effective detection of malaria in complex biological matrices and surveillance in the endemic region.

Breath Biomarkers of Pediatric Malaria: Reproducibility and Response to Antimalarial Therapy.

Many insect-borne pathogens appear to manipulate the odors of their hosts in ways that influence vector behaviors. In our prior work, we identified characteristic changes in volatile emissions of cultured Plasmodium falciparum parasites in vitro and during natural human falciparum malaria. In the current study, we prospectively evaluate the reproducibility of these findings in an independent cohort of children in Blantyre, Malawi. We enrolled febrile children under evaluation for malaria and collected breath from children with and without malaria, as well as healthy controls. Using gas chromatography/mass spectrometry, we characterized breath volatiles associated with malaria. By repeated sampling of children with malaria before and after antimalarial use, we determined how breath profiles respond to treatment. In addition, we investigated the stage-specificity of biomarkers through correlation with asexual and sexual-stage parasitemia. Our data provide robust evidence that P. falciparum infection leads to specific, reproducible changes in breath compounds. While no individual compound served as an adequate classifier in isolation, selected volatiles together yielded high sensitivity for diagnosis of malaria. Overall, the results of our predictive models suggest the presence of volatile signatures that reproducibly predict malaria infection status and determine response to therapy, even in cases of low parasitemia.

Rapid diagnosis and prognosis of malaria infection using a microfluidic point-of-care immunoassay

Malaria is a major cause of illness and death worldwide. Rapid diagnostic tests are the most widely used tool for detecting malaria infection, however, they only provide binary results and lack the sensitivity needed to detect many asymptomatic infections. Molecular assays for quantifying malaria biomarkers offer higher detection sensitivity, however, they are time-consuming, and require expert training and expensive equipment, making them unsuitable for use in most of Africa. To address the need for simple, accurate and field-deployable malaria diagnostic tests, we have developed a microfluidic point-of-care (mPOC) immunoassay for rapid quantification of Plasmodium falciparum histidine-rich protein 2 (PfHRP2), a malaria parasite biomarker, in whole blood. This device features two diagnostic modes for detecting PfHRP2 at low (100's pg/mL) and high (1,000's ng/mL) concentrations, making it useful for multiple diagnostic applications, including the detection of asymptomatic infection, prediction of disease outcomes and diagnosis of cerebral malaria. Measurements of PfHRP2 in blood samples from malaria patients demonstrates that this platform offers similar accuracy as an ultra-sensitive PfHRP2 enzyme-linked immunosorbent assay (ELISA) test, while being 12× faster and simpler to use. This mPOC immunoassay can be deployed in rural health centers to assist clinicians in diagnosing and triaging malaria patients, ultimately improving patient outcomes.

Hematin anhydride (β-hematin): An analogue to malaria pigment hemozoin possesses nonlinearity

Hematin anhydride (β-hematin), the synthetic analogue of the malaria pigment, “hemozoin”, is a heme dimer produced by reciprocal covalent bonds among carboxylic acid groups on the protoporphyrin-IX ring and the iron atom present in the two adjacent heme molecules. Hemozoin is a disposal product formed from the digestion of hemoglobin present in the red blood cells infected with hematophagous malaria parasites. Besides, as the parasites invade red blood cells, hemozoin crystals are eventually released into the bloodstream, where they accumulate over time in tissues. Severe malaria infection leads to significant dysfunction in vital organs such as the liver, spleen, and brain in part due to the autoimmune response to the excessive accumulation of hemozoin in these tissues. Also, the amount of these crystals in the vasculature correlates with disease progression. Thus, hemozoin is a unique indicator of infection used as a malaria biomarker and hence, used as a target for the development of antimalarial drugs. Hence, exploring various properties of hemozoin is extremely useful in the direction of diagnosis and cure. The present study focuses on finding one of the unknown properties of β-hematin in physiological conditions by using the Z-scan technique, which is simple, sensitive, and economical. It is observed that hemozoin possesses one of the unique material properties, i.e., nonlinearity with a detection limit of ∼ 15 µM. The self-defocusing action causes β-hematin to exhibit negative refractive nonlinearity. The observed data is analyzed with a thermal lensing model. We strongly believe that our simple and reliable approach to probing the nonlinearity of β-hematin will provide fresh opportunities for malaria diagnostics & cure in the near future.

Highly sensitive detection of malaria biomarker through matching channel and gate capacitance of integrated organic electrochemical transistors

Organic electrochemical transistors (OECTs) possess versatile advantages for biochemical and electrophysiological applications due to electrochemical gating and ion-to-electron conversion capability. Although OECTs have been successfully applied for biochemical sensing, the effect of relative capacitance for specific sensing events is still unclear. In the present work, we design integrated interdigitated OECTs (iOECTs) with on-plane gold gate and different channel geometries for point-of-care diagnosis of malaria using aptamer as receptor. The transconductance of the iOECTs gated with micro-size gold electrodes decreased with increasing the channel thicknesses, especially for devices with large channel areas, which is inconsistent with devices gated by typical Ag/AgCl electrodes, attributing to the limited gating efficiency of the micro-size gate electrode. The capacitance of gate electrode was heavily suppressed by receptors but increased with the incubation of targets. In addition, the integrated iOECTs with thin channels exhibited superior sensitivity for malaria detection with the detection limit as low as 3.2 aM, much lower than their thick channel counterpart and other state-of-the-art biosensors. These deviations could be caused by the high relative capacitances, with respect to the gate and channel capacitance (Cg/Cch), resulting in a high gate potential drop over the organic channel and thus entirely gating on the thin channel device. These findings provide guidance to optimize the geometry of OECT devices with on-chip integrated gates and the fabrication of miniaturized OECTs for biosensing applications.

Expression, Purification, and Characterization of Plasmodium vivax Lactate Dehydrogenase from Bacteria without Codon Optimization.

Plasmodium vivax is the most widespread cause of malaria, especially in subtropical and temperate regions such as Asia-Pacific and America. P. vivax lactate dehydrogenase (PvLDH), an essential enzyme in the glycolytic pathway, is required for the development and reproduction of the parasite. Thus, LDH from these parasites has garnered attention as a diagnostic biomarker for malaria and as a potential molecular target for developing antimalarial drugs. In this study, we prepared a transformed Escherichia coli strain for the overexpression of PvLDH without codon optimization. We introduced this recombinant plasmid DNA prepared by insertion of the PvLDH gene in the pET-21a(+) expression vector, into the Rosetta(DE3), an E. coli strain suitable for eukaryotic protein expression. The time, temperature, and inducer concentration for PvLDH expression from this E. coli Rosetta(DE3), containing the original PvLDH gene, were optimized. We obtained PvLDH with a 31.0 mg/L yield and high purity (>95%) from this Rosetta(DE3) strain. The purified protein was characterized structurally and functionally. The PvLDH expressed and purified from transformed bacteria without codon optimization was successfully demonstrated to exhibit its potential tetramer structure and enzyme activity. These findings are expected to provide valuable insights for research on infectious diseases, metabolism, diagnostics, and therapeutics for malaria caused by P. vivax.

Does food biodiversity protect against malnutrition and favour the resilience to climate change-related events in Amazon Indigenous communities? A protocol for a mixed methods study

Background: Undernutrition is projected to be a major consequence of climate change. Biodiversity could enhance climate change resilience by improving nutritional outcomes and providing healthy food resources during and/or after climate-related events. For Indigenous populations who currently base their diet on local biodiversity, rapid climate changes may affect their ability to produce, access or gather food and consequently impact their nutritional status. There is a knowledge gap regarding whether nutritional status among Indigenous populations is better among those who consume a diet with greater biodiversity than those who have a diet with low biodiversity. Objective: This study aims to investigate the role of food biodiversity (FBD) in nutritional resilience to extreme flooding events of Shawi Amazon Indigenous adults living in Peruvian communities that have experienced extreme floods in the past five years. Methods: This study will use a mixed-method sequential explanatory design. The quantitative component includes a cross-sectional survey to assess the association between food biodiversity (FBD) and the prevalence of anaemia in adults aged 15 to 60 years old (n=365). Anaemia will be evaluated using blood hemoglobin and serum ferritin. FBD will be measured with a food frequency questionnaire and a 24-hour dietary recall. Soil-transmitted helminth infections, malaria, and inflammatory biomarkers will also be evaluated. Qualitative component will include a community-based participatory approach to investigate the role of FBD in the responses to extreme floods. Male (n=14) and female (n=14) participants, previously identified in the quantitative phase with high and low levels of FBD, will be invited to participate in a Photovoice activity and semi-structured interviews. A analytical framework for climate change resilience will be used to integrate the data. Discussion: Findings will be integrated to identify features of diet quality to suggest nutritional interventions that are resilient to changing climatic conditions in the Amazon and respect Indigenous views.

Relevance of Plasmodium falciparum Biomarkers in the Treatment and Control of Malaria

We report here a dual-ELISA method to measure Malaria biomarkers concurrently in the same aliquot of blood sample. A correlation between the parasite numbers and ELISA values determined and the figures were used to establish a standard graph. Thick blood smears prepared from spiked blood samples were also Giemsa stained and parasite density determined by microscopy (It was thereby possible to undertake an objective comparison between lactate dehydrogenase and histidine- rich- proteins levels assessed by ELISA and parasite density determined by microscopy from the same spiked aliquot). The presence of lactate dehydrogenase and histidine- rich- proteins is localized and visualized by fluorescent antibody techniques. Twenty-two patient blood samples were retrospectively analyzed for the levels of pLDH and HRPs and their level was quantified in each blood sample. Fresh blood samples from malaria patients seeking care at a healthcare facility in an endemic area were then collected and separated into plasma and infected red blood cells and parasitemia levels determined. The biomarkers which were released into plasma from circulating and sequestered parasites provide a more accurate picture and indicate degree of disease severity. Since lactate dehydrogenase is known to have a short half-life compared to the histidine-rich proteins, its plasma level is believed to reflect on the number of metabolically active parasites more accurately and more reliable indicator of clinical outcome. In contrast, histidine -rich proteins accumulate in plasma and measurable levels persist long after patients have been treated and parasites cleared from their system. Best practice may now call for a pre-eminent role for microscopy and PCR in plasmodium species confirmation, a continued role for newer histidine-rich protein assays in epidemiological studies and control, and the preferential use of parasite lactate dehydrogenase in the clinical management of the disease. Data presented here from spiked blood samples as well as naturally infected patient samples provide evidence that by splitting whole blood samples into RBC-pellet and plasma, determination of parasite biomarkers in split sample fractions gives a true picture of malaria parasites present in humans and this may improve the accuracy of models that attempt to predict parasite burdens more accurately, and so clinical correlations.

Abstract 2662: Identification of next-generation biomarkers of Vivax Malaria and understanding disease severity using proteomics

Abstract 2662: Identification of next-generation biomarkers of Vivax Malaria and understanding disease severity using proteomics

Current Status and Future Perspectives of Lactate Dehydrogenase Detection and Medical Implications: A Review.

The demand for glucose uptake and the accompanying enhanced glycolytic energy metabolism is one of the most important features of cancer cells. Unlike the aerobic metabolic pathway in normal cells, the large amount of pyruvate produced by the dramatic increase of glycolysis in cancer cells needs to be converted to lactate in the cytoplasm, which cannot be done without a large amount of lactate dehydrogenase (LDH). This explains why elevated serum LDH concentrations are usually seen in cancer patient populations. LDH not only correlates with clinical prognostic survival indicators, but also guides subsequent drug therapy. Besides their role in cancers, LDH is also a biomarker for malaria and other diseases. Therefore, it is urgent to develop methods for sensitive and convenient LDH detection. Here, this review systematically summarizes the clinical impact of lactate dehydrogenase detection and principles for LDH detection. The advantages as well as limitations of different detection methods and the future trends for LDH detection were also discussed.

Does food biodiversity protect against malnutrition and favour the resilience to climate change-related events in Amazon Indigenous communities? A protocol for a mixed methods study.

Background : Undernutrition is projected to be a major consequence of climate change. Biodiversity could enhance climate change resilience by improving nutritional outcomes and providing healthy food resources during and/or after climate-related events. For Indigenous populations who currently base their diet on local biodiversity, rapid climate changes may affect their ability to produce, access or gather food and consequently impact their nutritional status. There is a knowledge gap regarding whether nutritional status among Indigenous populations is better among those who consume a diet with greater biodiversity than those who have a diet with low biodiversity. Objective : This study aims to investigate the role of food biodiversity (FBD) in nutritional resilience to extreme flooding events of Shawi Amazon Indigenous adults living in Peruvian communities that have experienced extreme floods in the past five years. Methods : This study will use a mixed-method sequential explanatory design. The quantitative component includes a cross-sectional survey to assess the association between food biodiversity (FBD) and the prevalence of anaemia in adults aged 15 to 60 years old (n=365). Anaemia will be evaluated using blood hemoglobin and serum ferritin. FBD will be measured with a food frequency questionnaire and a 24-hour dietary recall. Soil-transmitted helminth infections, malaria, and inflammatory biomarkers will also be evaluated. The qualitative component will include a community-based participatory approach to investigate the role of FBD in the responses to extreme floods. Male (n=14) and female (n=14) participants, previously identified in the quantitative phase with high and low levels of FBD, will be invited to participate in a Photovoice activity and semi-structured interviews. A analytical framework for climate change resilience will be used to integrate the data. Discussion : Findings will be integrated to identify nutritional resilience indicators that can inform adaptative interventions to changing climatic conditions in the Amazon and that respect Indigenous worldviews.

Application of the aqueous two-phase system and nanozyme signal enhancement for the improved detection of Plasmodium lactate dehydrogenase in serum.

Malaria is an infectious disease that can cause severe sickness and death if not diagnosed and treated in a timely manner. The current gold standard technique for malaria diagnosis is microscopy, which requires a dedicated laboratory setting and trained personnel and can have a long time to result. These requirements can be alleviated using paper-based diagnostic devices that enable rapid and inexpensive diagnosis at the point of care, which can allow patients to receive treatment before their symptoms progress when used for early detection of diseases. The lateral-flow immunoassay (LFA) is one such device, but currently available LFAs are susceptible to false negative results caused by low parasite density. To improve sensitivity and detection, we utilized the aqueous two-phase system (ATPS) to concentrate and purify the sample, and nanozyme signal enhancement to increase the intensity of the visible signal on the test strip. We were able to achieve a limit of detection (LOD) of 0.01ng/mL for the malaria biomarker Plasmodium lactate dehydrogenase (pLDH) in human serum using a multi-step assay combining the LFA format with the ATPS and nanozyme signal enhancement.

Procalcitonin as a Candidate Biomarker for Malarial Infection and Severe Malaria: A Meta-Analysis.

Procalcitonin (PCT), as a marker of malaria severity, remains to be investigated. The present study collated and compared the levels of PCT between patients with severe malaria, uncomplicated malaria, and control participants to assess their role in predicting malaria infection and disease severity. The systematic review was registered at PROSPERO with registration number CRD42021297243. The search for relevant studies that reported PCT in patients with malaria was performed in PubMed, Scopus, and Web of Science. The following meta-analyses were conducted; (1) the pooled mean PCT levels in patients with severe and uncomplicated malaria, and (2) the pooled mean difference in PCT levels between patients with severe and uncomplicated malaria. Fifteen studies were included for qualitative and quantitative syntheses. The meta-analysis results show that the pooled mean PCT levels in patients with uncomplicated malaria were 3.92 ng/mL (95% CI: 2.26–5.58 ng/mL, I2: 96.5, five studies), whereas the pooled mean PCT levels in patients with severe malaria were 14.13 ng/mL (95% CI: 8.75–19.5 ng/mL, I2: 92.6, six studies). The meta-analysis showed that patients with severe malaria had an equal mean of PCT compared to those with uncomplicated malaria when the random-effects model was used (p: 0.055, weighted mean difference: 6.93, 95% CI: −0.16–14.02, I2: 84.6%, four studies). There were probable correlations between the level of parasitemia, immunity level, and possibly bacterial or other parasitic co-infection that could affect the PCT level among different clinical severities of malaria. Therefore, the PCT level alone does not seem to be a suitable biomarker to discriminate the severe/uncomplicated or infected/uninfected cases. Further studies should investigate the increased PCT levels in combination with other markers in association with malaria infection and severity.

Limited genetic diversity and expression profile of Plasmodium falciparum haem detoxification protein: a possible diagnostic target.

Haem detoxification protein (HDP) is a significant protein in the erythrocytic stage of the Plasmodium lifecycle. HDP could be of paramount interest as a diagnostic biomarker for accurate diagnosis of malaria. We thus explored HDP genetic variation, expression levels of HDP and immune response. Phylogenetic analysis was carried out using Pfhdp orthologues sequences of various Plasmodium species. Blood samples were collected from patients in central India. Pfhdp gene was amplified, and sequenced by sanger DNA sequencing. B-cell epitopes were identified in PfHDP using Bepipred Linear Epitope Prediction 2.0, and median-joining network was constructed using global PfHDP sequences. Pfhdp expression levels during erythrocytic stage were assessed using real-time qPCR at 4-h intervals. An IgG immune response against synthetic PfHDP peptides was analysed using ELISA. Phylogenetic analysis revealed the conserved nature of Pfhdp gene. Diversity analysis revealed one non-synonymous mutation (F91L) among all isolates. Neutrality tests indicated negative selection for Pfhdp gene. HDP was expressed throughout the erythrocytic cycle, and comparatively, high expression was observed in the late trophozoite and schizont stages. High IgG response against both peptides was observed, and no polymorphism was seen in any of the seven predicted B-cell epitopes. Findings of the present study indicate the possibility of HDP being exploited as a diagnostic biomarker for Plasmodium falciparum malaria after proteomic validation studies.

Delineating charge and capacitance transduction in system-integrated graphene-based BioFETs used as aptasensors for malaria detection

Despite significant eradication efforts, malaria remains a persistent infectious disease with high mortality due to the lack of efficient point-of-care (PoC) screening solutions required to manage low-density asymptomatic parasitemia. In response, we demonstrate a quantitative electrical biosensor based on system-integrated two-dimensional field-effect transistors (2DBioFETs) of reduced graphene oxide (rGO) as transducer for high sensitivity screening of the main malaria biomarker, Plasmodium falciparum lactate dehydrogenase (PfLDH). The 2DBioFETs were biofunctionalized with pyrene-modified 2008s aptamers as specific PfLDH receptors. While we systematically optimize biosensor interface for optimal performance, aptamer-protein transduction at 2DBioFETs is elucidated based on delineation of charge and capacitance in an updated analytical model for two-dimensional rGO/biofunctional layer/electrolyte (2DiBLE) interfaces. Our 2DBioFET-aptasensors display a limit-of-detection down to 0.78 fM (0.11 pg/mL), dynamic ranges over 9 orders of magnitude (subfemto to submicromolar), high sensitivity, and selectivity in human serum validating their diagnostic potential as rapid PoC tests for malarial management.

Review of Microdevices for Hemozoin-Based Malaria Detection.

Despite being preventable and treatable, malaria still puts almost half of the world’s population at risk. Thus, prompt, accurate and sensitive malaria diagnosis is crucial for disease control and elimination. Optical microscopy and immuno-rapid tests are the standard malaria diagnostic methods in the field. However, these are time-consuming and fail to detect low-level parasitemia. Biosensors and lab-on-a-chip devices, as reported to different applications, usually offer high sensitivity, specificity, and ease of use at the point of care. Thus, these can be explored as an alternative for malaria diagnosis. Alongside malaria infection inside the human red blood cells, parasites consume host hemoglobin generating the hemozoin crystal as a by-product. Hemozoin is produced in all parasite species either in symptomatic and asymptomatic individuals. Furthermore, hemozoin crystals are produced as the parasites invade the red blood cells and their content relates to disease progression. Hemozoin is, therefore, a unique indicator of infection, being used as a malaria biomarker. Herein, the so-far developed biosensors and lab-on-a-chip devices aiming for malaria detection by targeting hemozoin as a biomarker are reviewed and discussed to fulfil all the medical demands for malaria management towards elimination.

Bioinformatics Analysis on MicroRNAs that Modulate Significant Host Response Genes as Potential Biomarkers in Cerebral Malaria Infection

Introduction: Pathogenesis of severe malaria and cerebral malaria infection is a major problem that affects human body. Candidate genes related to the pathogenesis of malaria disease such as CD36, IFN-γ, TLR4, PRR15 may associate with host microRNA. The contribution of the malaria pathogenesis with host cytokine responses through the cerebral malaria infection may associate with significant miRNAs serving for biomarkers still remain unclear. Objectives: This study was focused on bioinformatics analysis of host target miRNAs response genes association with pathogenesis of malaria as a potential biomarker for development of severe malaria and cerebral malaria. Materials and methods: Two different bioinformatics tools including miRanda and Target Scan were used in the prediction of the host genes associated specifically with miRNA as potential biomarkers of malaria. Results: The prediction result using bioinformatics tools showed that miR-203a-3p.1, miR-146, miR-155-5p, miR-425-5p, miR-217, miR-153-3p, miR-455-3p.2, miR-223, miR-143-3p, miR-146-5p, miR- 216a-5p were able to regulate host genes during the development of severe malaria and cerebral malaria. Conclusion: The bioinformatics analysis of the host microRNA as potential biomarkers from selected gene CD36, TLR4, IFN-γ and PRR15 using bioinformatics tools designated that associated with definite miRNAs such as miR-146 and miR-155. The circulating microRNAs associated with panels of significant host genes should be further investigated.

PAD4 controls chemoattractant production and neutrophil trafficking in malaria.

Peptidylarginine deiminase 4 (PAD4) is a key regulator of inflammation but its function in infections remains incompletely understood. We investigate PAD4 in the context of malaria and demonstrate a role in regulation of immune cell trafficking and chemokine production. PAD4 regulates liver immunopathology by promoting neutrophil trafficking in a Plasmodium chabaudi mouse malaria model. In human macrophages, PAD4 regulates expression of CXCL chemokines in response to stimulation with TLR ligands and P. falciparum. Using patient samples, we show that CXCL1 may be a biomarker for severe malaria. PAD4 inhibition promotes disease tolerance and may represent a therapeutic avenue in malaria.

Characterization and utility of immobilized metal affinity-functionalized cellulose membranes for point-of-care malaria diagnostics

Immobilized metal affinity chromatography (IMAC) is a well-established technique for protein separation and purification. IMAC has been previously utilized to capture the malaria biomarker histidine-rich protein 2 (HRP2) from blood, enhancing the sensitivity of field-appropriate diagnostic tools such as lateral flow assays. However, little work has been done to translate this technique to a truly field-usable design. In this study, IMAC-functionalized cellulose membranes are created and characterized fully for future use in applied malaria diagnostics. IMAC-functionalized cellulose membranes were investigated across a range of cellulose substrates, IMAC ligands, and divalent transition metals before use in a capture and elution flowthrough workflow. Following characterization and optimization, it was found that iminodiacetic acid bound to Zn(II) was the most promising ligand–metal pair, with three available coordination sites and a molar loading capacity of 57.7 μmol of metal/cm3 of cellulose. Using these parameters, more than 99% of HRP2 was captured from a large-volume lysed blood sample in a simple flow-through assay and 89% of the captured protein was eluted from the membrane using the chelating compound ethylenediaminetetraacetic acid. Use of this enhancement protocol on an in-house HRP2 lateral flow assay (LFA) yielded a limit of detection of 7 parasites/μL, a 15.8x enhancement factor compared to traditional LFA methods.

Towards malaria field diagnosis based on surface-enhanced Raman scattering with on-chip sample preparation and near-analyte nanoparticle synthesis

We report a chip based on surface-enhanced Raman scattering (SERS) developed towards malaria field diagnosis. Only a mixture of 10-μl water and 10-μl blood is required as the sample input to the chip. Water is the only lysing agent to hemolyze blood cells while keeping the malaria biomarkers, hemozoin biocrystals, at locally high concentrations within parasites and/or their vacuoles. Then, SERS-active silver nanoparticles are synthesized on site near hemozoin in these concentrated regions when the blood/water mixture flows through and dissolves dried chemical patches that are earlier deposited inside the channel, which subsequently arrives at the detection region for SERS measurements. It should be highlighted that the procedure can be accomplished without a laboratory requirement and the risk of exposure to hazardous chemicals. Additionally, raw chemicals deposited inside the chip are chemically more stable than those readymade SERS substrates, thus the shelf life of the chip can be much longer. Furthermore, the chip yields analytical enhancement factor values ranging from 5.4 × 103 to 1.9 × 106 that are comparable to other ready-made SERS substrates in the literature. This strategy is capable of quantifying hemozoin concentrations in malaria infected human blood with a root-mean-square error of prediction of 0.3 μM, and a detection limit of 0.0025 % parasitemia level for parasites in the ring stage (equivalent to 125 parasites/μl) with a room of extra enhancement by switching the laser to a more suitable wavelength. These results show the feasibility to exploit this cost-effective yet highly sensitive SERS-based technique for malaria field diagnosis.