Ultrasensitive electrochemical genosensors for species-specific diagnosis of malaria.

The absence of reliable species-specific diagnostic tools for malaria at point-of-care (POC) remains a major setback towards effective disease management. This is partly due to the limited sensitivity and specificity of the current malaria POC diagnostic kits especially in cases of low-density parasitaemia and mixed species infections. In this study, we describe the first label-free DNA-based genosensors based on electrochemical impedance spectroscopy (EIS) for species-specific detection of P. falciparum, P. malariae and P. ovale. The limits of detection (LOD) for the three species-specific genosensors were down in attomolar concentrations ranging from 18.7 aM to 43.6 aM, which is below the detection limits of previously reported malaria genosensors. More importantly, the diagnostic performance of the three genosensors were compared to quantitative real-time polymerase chain reaction (qPCR) assays using purified genomic DNA and the paired whole blood lysates from clinical samples. Remarkably, all the qPCR-positive purified genomic DNA samples were correctly identified by the genosensors indicating 100% sensitivity for each of the three malaria species. The specificities of the three genosensors ranged from 66.7% to 100.0% with a Therapeutic Turnaround Time (TTAT) within 30 min, which is comparable to the TTAT of current POC diagnostic tools for malaria. This work represents a significant step towards the development of accurate and rapid species-specific nucleic acid-based toolkits for the diagnosis of malaria at the POC.

Development of Cooperative Primer-Based Real-Time PCR Assays for the Detection of Plasmodium malariae and Plasmodium ovale.

Plasmodium malariae and Plasmodium ovale are increasingly gaining public health attention as the global transmission of falciparum malaria is decreasing. However, the absence of reliable Plasmodium species-specific detection tools has hampered accurate diagnosis of these minor Plasmodium species. In this study, SYBR Green-based real-time PCR assays were developed for the detection of P. malariae and P. ovale using cooperative primers that significantly limit the formation and propagation of primers-dimers. Both the P. malariae and P. ovale cooperative primer-based assays had at least 10-fold lower detection limit compared with the corresponding conventional primer-based assays. More important, the cooperative primer-based assays were evaluated in a cross-sectional study using 560 samples obtained from two health facilities in Ghana. The prevalence rates of P. malariae and P. ovale among the combined study population were 18.6% (104/560) and 5.5% (31/560), respectively. Among the Plasmodium-positive cases, P. malariae and P. ovale mono-infections were 3.6% (18/499) and 1.0% (5/499), respectively, with the remaining being co-infections with Plasmodium falciparum. The study demonstrates the public health importance of including detection tools with lower detection limits in routine diagnosis and surveillance of nonfalciparum species. This will be necessary for comprehensively assessing the effectiveness of malaria interventions and control measures aimed toward global malaria elimination.

Functional Molecular Interfaces for Impedance-Based Diagnostics.

In seeking to develop and optimize reagentless electroanalytical assays, a consideration of the transducing interface features lies key to any subsequent sensitivity and selectivity. This review briefly summarizes some of the most commonly used receptive interfaces that have been employed within the development of impedimetric molecular sensors. We discuss the use of high surface area carbon, nanoparticles, and a range of bioreceptors that can subsequently be integrated. The review spans the most commonly utilized biorecognition elements, such as antibodies, antibody fragments, aptamers, and nucleic acids, and touches on some novel emerging alternatives such as nanofragments, molecularly imprinted polymers, and bacteriophages. Reference is made to the immobilization chemistries available along with a consideration of both optimal packing density and recognition probe orientation. We also discuss assay-relevant mechanistic details and applications in real sample analysis.

Ultrasensitive Impedimetric Immunosensor for the Detection of C-Reactive Protein in Blood at Surface-Initiated-Reversible Addition-Fragmentation Chain Transfer Generated Poly(2-hydroxyethyl methacrylate) Brushes.

The reversible addition-fragmentation chain transfer (RAFT) polymerization of 2-hydroxethyl methacrylate (HEMA) from a surface confined, dithio-tethered, chain transfer agent (CTA) enables the preparation of electrode-tethered poly(2-hydroxyethyl methacrylate) (pHEMA) brushes of well-defined thickness with convenience and exceptionally high interfacial impedimetric baseline stability. The subsequent covalent integration of antibodies generates interfaces of very high target recognition specificity, ultimately enabling femtomolar levels of quantification of C-reactive protein (CRP) and recovery in spiked serum samples of ∼98%. When combined with the intrinsic scalability of the reagentless electrochemical impedance spectroscopy (EIS) platform, and the innate high levels of polymer tuneability and control, we believe this represents a valuable contribution to the diagnostic toolbox.

Recent Advances in the Development of Biosensors for Malaria Diagnosis.

The impact of malaria on global health has continually prompted the need to develop more effective diagnostic strategies that could overcome deficiencies in accurate and early detection. In this review, we examine the various biosensor-based methods for malaria diagnostic biomarkers, namely; Plasmodium falciparum histidine-rich protein 2 (PfHRP-2), parasite lactate dehydrogenase (pLDH), aldolase, glutamate dehydrogenase (GDH), and the biocrystal hemozoin. The models that demonstrate a potential for field application have been discussed, looking at the fabrication and analytical performance characteristics, including (but not exclusively limited to): response time, sensitivity, detection limit, linear range, and storage stability, which are first summarized in a tabular form and then described in detail. The conclusion summarizes the state-of-the-art technologies applied in the field, the current challenges and the emerging prospects for malaria biosensors.

Enzyme-based amperometric galactose biosensors: a review.

This review (with 35 references) summarizes the various strategies used in biosensors for galactose, and their analytical performance. A brief comparison of the enzyme immobilization methods employed and the analytical performance characteristics of a range of galactose biosensors are first summarized in tabular form and then described in detail. Selected examples have been included to demonstrate the various applications of these biosensors to real samples. Following an introduction into the field that covers the significance of sensing galactose in various fields, the review covers biosensors based on the use of galactose oxidase, with a discussion of methods for their immobilization (via cross-linking, adsorption, covalent bonding and entrapment). This is followed by a short section on biosensors based on the use of galactose dehydrogenase. The conclusion section summarizes the state of the art and addresses current challenges. Graphical abstractFabrication of a disposable screen-printed (a) electrochemical galactose biosensor (b) for real sample analysis and a dummy biosensor

Recent Progress in the Development of Diagnostic Tests for Malaria.

The impact of malaria on global health has continually prompted the need to develop effective diagnostic strategies. In malaria endemic regions, routine diagnosis is hampered by technical and infrastructural challenges to laboratories. These laboratories lack standard facilities, expertise or diagnostic supplies; thus, therapy is administered based on clinical or self-diagnosis. There is the need for accurate diagnosis of malaria due to the continuous increase in the cost of medication, and the emergence and spread of drug resistant strains. However, the widely utilized Giemsa-stained microscopy and immunochromatographic tests for malaria are liable to several drawbacks, including inadequate sensitivity and false-positive outcomes. Alternative methods that offer improvements in performance are either expensive, have longer turnaround time or require a level of expertise that makes them unsuitable for point-of-care (POC) applications. These gaps necessitate exploration of more efficient detection techniques with the potential of POC applications, especially in resource-limited settings. This minireview discusses some of the recent trends and new approaches that are seeking to improve the clinical diagnosis of malaria.

A Disposable Amperometric Sensor Based on High-Performance PEDOT:PSS/Ionic Liquid Nanocomposite Thin Film-Modified Screen-Printed Electrode for the Analysis of Catechol in Natural Water Samples.

A conducting polymer-based composite material of poly(3,4-ethylenedioxythiophene) (PEDOT): poly(4-styrenesulfonate) (PSS) doped with different percentages of a room temperature ionic liquid (IL), 1-ethyl-3-methylimidazolium tetrafluoroborate ([EMIM][BF4]), was prepared and a very small amount of the composite (2.0 µL) was drop-coated on the working area of a screen-printed carbon electrode (SPCE). The SPCE, modified with PEDOT:PSS/IL composite thin-film, was characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), scanning electron microscopy (SEM), profilometry and sessile contact angle measurements. The prepared PEDOT:PSS/IL composite thin-film exhibited a nano-porous microstructure and was found to be highly stable and conductive with enhanced electrocatalytic properties towards catechol, a priority pollutant. The linear working range for catechol was found to be 0.1 µM-330.0 µM with a sensitivity of 18.2 mA·mM·cm-2 and a calculated limit of detection (based on 3× the baseline noise) of 23.7 µM. When the PEDOT:PSS/IL/SPCE sensor was used in conjunction with amperometry in stirred solution for the analysis of natural water samples, the precision values obtained on spiked samples (20.0 µM catechol added) (n = 3) were 0.18% and 0.32%, respectively, with recovery values that were well over 99.0%.

Investigating the Influence of Temperature on the Kaolinite-Base Synthesis of Zeolite and Urease Immobilization for the Potential Fabrication of Electrochemical Urea Biosensors.

Temperature-dependent zeolite synthesis has revealed a unique surface morphology, surface area and pore size which influence the immobilization of urease on gold electrode supports for biosensor fabrication. XRD characterization has identified zeolite X (Na) at all crystallization temperatures tested. However, N2 adsorption and desorption results showed a pore size and pore volume of zeolite X (Na) 60 °C, zeolite X (Na) 70 °C and zeolite X (Na) 90 °C to range from 1.92 nm to 2.45 nm and 0.012 cm³/g to 0.061 cm³/g, respectively, with no significant differences. The specific surface area of zeolite X (Na) at 60, 70 and 90 °C was 64 m²/g, 67 m²/g and 113 m²/g, respectively. The pore size, specific surface area and pore volumes of zeolite X (Na) 80 °C and zeolite X (Na) 100 °C were dramatically increased to 4.21 nm, 295 m²/g, 0.762 cm³/g and 4.92 nm, 389 m²/g, 0.837 cm³/g, in that order. The analytical performance of adsorbed urease on zeolite X (Na) surface was also investigated using cyclic voltammetry measurements, and the results showed distinct cathodic and anodic peaks by zeolite X (Na) 80 °C and zeolite X (Na) 100 °C. These zeolites' molar conductance was measured as a function of urea concentration and gave an average polynomial regression fit of 0.948. The findings in this study suggest that certain physicochemical properties, such as crystallization temperature and pH, are critical parameters for improving the morphological properties of zeolites synthesized from natural sources for various biomedical applications.

Immunochemical Assays and Nucleic-Acid Detection Techniques for Clinical Diagnosis of Prostate Cancer.

Prostate cancer (PCa) is a significant cause of morbidity and mortality and the most common cancer in men in Europe, North America, and some parts of Africa. The established methods for detecting PCa are normally based on tests using Prostate Specific Antigen (PSA) in blood, Prostate cancer antigen 3 (PCA3) in urine and tissue Alpha-methylacyl-CoA racemase (AMACR) as tumour markers in patient samples. Prior to the introduction of PSA in clinics, prostatic acid phosphatase (PAP) was the most widely used biomarker. An early diagnosis of PCa through the detection of these biomarkers requires the availability of simple, reliable, cost-effective and robust techniques. Immunoassays and nucleic acid detection techniques have experienced unprecedented growth in recent years and seem to be the most promising analytical tools. This growth has been driven in part by the surge in demand for near-patient-testing systems in clinical diagnosis. This article reviews immunochemical assays, and nucleic-acid detection techniques that have been used to clinically diagnose PCa.

Simultaneous electrochemical determination of dopamine and 5-hydroxyindoleacetic acid in urine using a screen-printed graphite electrode modified with gold nanoparticles.

The authors describe a disposable screen-printed graphite electrode (SPGE) modified with gold nanoparticles (AuNPs). The electrode, designated as AuNPs-SPGE, was characterized by cyclic voltammetry and electrochemical impedance spectroscopy. The AuNPs-SPGE combines the electrochemical features of graphite and the disposability of screen-printed electrodes. It displays excellent electrocatalytic activity towards dopamine (DA) and 5-hydroxyindoleacetic acid (5-HIAA). Two well-defined, sharp, and fully resolved voltammetric peaks (at 525 mV for DA and at 415 mV for 5-HIAA, both vs. Ag/AgCl) were found. Square wave voltammetry was used to simultaneously determine DA and 5-HIAA in mixtures and in urine. The linear working range extends from 0.1 to 120.0 µM for both DA and 5-HIAA, and the limits of detection (based on 3× the baseline noise) are 14.0 and 5.7 nM, respectively. The fabrication method for the AuNPs-SPGE is highly reproducible. The performance of the AuNPs-SPGE was evaluated by analyzing spiked human urine, and the recoveries were found to be well over 94.0 % for both compounds. These results indicate that the AuNPs-SPGE represents a highly selective and sensitive sensor for simultaneous determination of DA and 5-HIAA in urine. Graphical Abstract Fabrication, characterization and electrochemical behavior of gold nanoparticles modified screen-printed graphite electrode towards dopamine and 5-hydroxyindoleacetic acid in human urine.

Development of an amperometric screen-printed galactose biosensor for serum analysis.

The development of a disposable amperometric biosensor for the measurement of circulating galactose in serum is described. The biosensor comprises a screen-printed carbon electrode (SPCE), incorporating the electrocatalyst cobalt phthalocyanine (CoPC), which is covered by a permselective cellulose acetate (CA) membrane and a layer of immobilized galactose oxidase (GALOX). The optimal response of the biosensor, designated as GALOX-CA-CoPC-SPCE, was obtained by systematically examining the effects of enzyme loading, temperature, pH, and buffer strength. The optimal performance of the biosensor occurred with 2U of GALOX, at 35°C, using 50mM phosphate buffer solution (pH 7.0). The sensitivity was 7.00µAmM(-1)cm(-2) and the linear range from 0.1 to 25mM with a calculated limit of detection (LOD) of 0.02mM; this concentration range and LOD are appropriate to diagnose galactosemia, i.e., concentrations >1.1mM in infants. When the biosensor was used in conjunction with amperometry in stirred solution for the analysis of serum, the precision values obtained on unspiked (endogenous level of 0.153mM) and spiked serum (1mM added) (n=6) were 1.10% and 0.11%, respectively, with a calculated recovery of 99.9%.