sensors Review Recent Advances in the Development of Biosensors for Malaria Diagnosis Francis D. Krampa 1,2,* , Yaw Aniweh 1 , Prosper Kanyong 1,3 and Gordon A. Awandare 1,2 1 West African Centre for Cell Biology of Infectious Pathogens (WACCBIP), University of Ghana, P.O. Box LG 25, Legon, Accra, Ghana; aniweh@gmail.com (Y.A.); p.kanyong@waccbip.org (P.K.); gawandare@ug.edu.gh (G.A.A.) 2 Department of Biochemistry, Cell & Molecular Biology, University of Ghana, P.O. Box LG 54, Legon, Accra, Ghana 3 Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QZ, UK * Correspondence: fkrampa@gmail.com  Received: 7 October 2019; Accepted: 24 December 2019; Published: 1 February 2020  Abstract: 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. Keywords: malaria biomarkers; biosensors; clinical diagnosis; medical devices; biosensing 1. Introduction Malaria remains an important parasitic human disease globally, which is transmitted via the bite of female Anopheles mosquitoes. The greatest burden of the disease is in the tropical and subtropical regions of the world [1,2]. The disease causes high economic burden to the countries that are endemic, mostly, developing countries. The etiologic agent is an Apicomplexan protozoan of the genus Plasmodium. Six species of this genus, namely, Plasmodium falciparum, Plasmodium malariae, Plasmodium knowlesi, Plasmodium ovale (P. ovale curtisi and P.ovale wallikeri), Plasmodium cynomolgi and Plasmodium vivax are known to cause infection in humans. As the World Health Organization (WHO) sets the goal for malaria elimination by 2030 [3], the aim can only be achieved when all cases are accurately diagnosed and treated appropriately. Some of the endemic communities still lack access to routine testing in suspected cases. For example, in 2018, only 74% of patients suspected to have malaria, excluding undocumented cases, had access to diagnostic tests in public health facilities [2]. A total of 228 million cases were recorded worldwide during this period out of which 405,000 mortalities occurred [4]. Different control strategies have been effective, but limited by ineffective early diagnostic tools for detection, especially, at low parasitemia and surveillance in low-transmission settings. The ability to detect asymptomatic individuals will greatly impact on transmission dynamics, malaria control, and possibly towards elimination. Diagnostic testing may help health service providers to further investigate other aetiologies of febrile illnesses; prevent severe disease and probable death; reduce the presumptive use of antimalarial drugs and associated side-effects; and mitigate against the rapid Sensors 2020, 20, 799; doi:10.3390/s20030799 www.mdpi.com/journal/sensors Sensors 2020, 20, x FOR PEER REVIEW 2 of 21 emergence and spread of drug resistance. It could also reduce the pool of individuals who can contribute to malaria transmission [5]. To date, many technologies have attempted to circumvent the challenges in malaria diagnostics with technologies that address point-of-care needs and early stage asymptomatic detection. In this reviewSe,n wsores 2fi0r2s0t, 2c0o,m79p9 rehensively summarize the biomarkers targeted during the course of malaria 2 of 21 with emphasis on the importance of sensitive early detection. Next, we provide an overview of the recente amdevragnecnecse ina nbdiossepnrseoard teocfhdnrouloggrieess ifsotar nthcee .deItteccotiuolnd oaf ltshoe rmedoustc etarthgeetepdo oblioomf ainrkdeivrsi,d fuoaclussiwngh o can on: decvoenltorpibmuetentt,o amnaallyartiicaatlr apnesrmfoirsmsiaonnc[e5s],. and suitability for point of care testing. The prevailing challenges Taondd aftuet,umrea nouyttleocohkn oofl othgeie us shea ovfe tahtetesem tpetcehdnotolocgiriecsu minv tehnet ftiheledc ahrael laelnsog ehsiginhlmigahlaterdia. diagnostics 2. Parwasiitthe tDecehvneloolopgmieesntth iant Hadudmraesnss,p Boiionmt-oafr-kcearrse, naenedd Ds aiangdneoasrilsy stage asymptomatic detection. In this review, we first comprehensively summarize the biomarkers targeted during the course of malaria Twheit hdeevmelpohpamsiesnotanl tchyecliem opfo Prltaasnmcoedoiufmse snpseitciivees tehaartly indfectet chtuiomn.anNse ixs tb, rwieeflpyr iollvuisdteraatnedo ivne rFvigieuwreo f the 1 [6]. Trehcee cnytcaled vbaengicness winitbhi othsen isnojerctteiochnn oofl sopgoiersozforittehse indteot ethctei ohnosotf’st hciercmuolasttiotanr gbeyt eadn ibniofemctaerdk eferms, afolec using Anophoenle:s dmevoseqloupitmo.e nTth,ea nspaloyrtoiczaolitpeesr ftohremn atnarcgeest, aand seunittearb ihlietypaftoorcpytoeisn twohfecraer ethtesyt imngu.ltTiphley parnedva iling differecnhtailalteen ginetsoa nmderfuoztuorieteos.u Ttlhooisk sotaf gthee ouf stehoef pthaerasesittec lhifneo lcoygciles iisn ktnhoewfienl dasa rperael-seoryhtihgrholcigyhtitce. dI.n infections involving P. vivax and P. ovale, dormant forms of the liver stage, called hypnozoites may persis2t .inP athraes liitveeDr [e7v]e alonpdm caeunstei nreHlaupmsea onfs t,hBei oinmfeacrtkioenrs, ,tahnerdeDbyi amgnakoisnisg it difficult to eradicate. The pre-erythroTchyeticd esvtealgoep mise netasslecnytcialel oinf Ptlhasem oedstiuabmlisshpmeceinest tohfa tminafleacrtiah uinmfeacntsioins. bTriheifls ysitlalugest raist ed in asympFtiogmuraeti1c, [h6o].weTvheer,c iyt cilse dbifefgiciunlst wfoirt hditahgenoinsjteicc ttiooonlso tfos dpeotreoczt osipteosroiznotoitetsh ebehcoasuts’se hcierpcuatloactiyotnesb y an invasiionnfe occtceudrsf ewmitahlien A30n–o4p5h emleisn mafotesrq supitoor.ozToihtees saproe rionzoociutleastetdh ebny tthareg ientfeacntedd emnotesrquhietop a[8to,9c]y. tTehsisw here short ttihmeye amnudl ltoipwly naunmdbderisff eorfe snptoiartoezoinitteos minejercotzeodi tleesa.veTsh liitstlset atigmeeo ffotrh tehepira rdaestietectiloifne. cSyocmlee iesffkonrtosw n as in findpirne-ge rbyitohmroacrykteircs. Ifnori ndfeecteticotniosni novfo elvairnlyg Pli.vveirv asxtaagned oPr. otvhael ed, odromrmanatn tfoformrm hsaovfet hiedelinvteifriestda gteh,ec alled Plasmohdyipunmo lziovietre-ssmpeacyifpice prsriostteiinnt 2h e(LliIvSePr2[)7 []1a0n,1d1c].a Tuhsee rseplaoprosezooifteth seuirnfafeccet icoinrc,uthmesrpeboyromzoaiktien pgriottdeiiffinsc ult to (CSP),e rwadhiiccaht ef.uTnhcetiponres- etroy tihnrtoecryatcitc wstaitghe riseceespsetonrtisa loinn tthhee ehsteapbaltisohcmyteen, thoafsm aalslaor ibaeiennfe ctatirogne.teTdh isfosrt age is diagnaossytimc ppototemnatitaicl ,[1h2o]w. ever, it is difficult for diagnostic tools to detect sporozoites because hepatocytes Tihnev aesriyotnhroocccyutircs swtaigthe ionf 3in0f–e4c5tiomni nbeagftiners swphoerno zmoietreoszaorieteisn roecleualasetedd frboymt htheei ninfevcatdeed rmedo sbqlouoitdo [8,9]. cells (RThBiCsss)h aonrdt tgimroewa fnrdomlo twhen ruinmgbse tros torfopsphoorzoozioteiste asnidn jsecchteizdolnetasv setsaglietstl oeft idmeveefloorptmheeinrtd. Sectehciztioonnt.s Some egresse fftoo rrteslienasfien dminegrobziooimteas rtkhearts fcoorndtientuecet itohne ocfyecalriclyall iavseerxsutaagl ecyocrlteh. eInd otrhme apnrtofcoersms, hsaovmeei doefn tthifiee d the parasiPtelass mdiofdfeiuremntliiavteer -isnptoec gifiacmpertootceyitnes2 t(oL IbSePg2i)n[ t1h0e,1 s1e].xuTahle pshpaosreo zoof itthees ulirffea ccyecclier.c Tumhes pgaomroeztooicteytperso teins are tak(CenSP u)p, w bhyi cfehmfuanlec tAionnospthoeinletse rmacotswquitihtoreesc edputorirnsgo nbltohoedh mepeaatlo. cTyhteis, hsuasbaselsqoubeenetnlyt adregveetelodpfso rind tihagen ostic midgupto tthenrotiuagl h[1 t2o]. ookinete and their transition into the salivary glands as sporozoites ready to be injected during blood meal to initiate infection in humans. FigureF 1ig. Dureev1e.loDpemveenlotaplm cyencltea locfy hculemoafnh PulmasamnoPdiluasmm sopdieucmiess p(reecdieessi(grendeeds figronmed SfcrhoemrfS ecth aelr. f2e0t0a8l). i2n0 a0 8) in a mammmalaimanm haolsiat nanhdo stthaen sdtrtahteegsitersa tuesgeides inu sdeedteicntidnegt epcatirnagsiptea srpaseictiefiscp mecaifirkcemrsa. rkers. The erythrocytic stage of infection begins when merozoites released from the invade red blood cells (RBCs) and grow from the rings to trophozoites and schizonts stages of development. Schizonts egress to release merozoites that continue the cyclical asexual cycle. In the process, some of the parasites Sensors 2020, 20, 799 3 of 21 differentiate into gametocytes to begin the sexual phase of the life cycle. The gametocytes are taken up by female Anopheles mosquitoes during blood meal. This subsequently develops in the midgut through to ookinete and their transition into the salivary glands as sporozoites ready to be injected during blood meal to initiate infection in humans. Various parasite detection methods have been employed over the years in diagnosing malaria cases. It is ideal to detect infection at the erythrocytic stage because of exponentially elevated parasite numbers, abundance of nucleic acid markers or the production of soluble antigenic proteins that can illicit immune responses. Using a microscope and efficient staining of peripheral blood, popularly Giemsa-stained thick and thin blood films, parasitized RBCs can be visualized and the different parasite species distinguished morphologically in: ring, trophozoite, schizont, and gametocyte. Although microscopy offers advantages such as good sensitivity and the capacity to determine parasitemia and the type of species, it is time-consuming and requires a highly skilled microscopist. Cases are sometimes misdiagnosed or undetected due to poor sensitivity at low parasitemia, which lead to inappropriate and/or delayed treatment [13,14]. These limitations associated with the routine use of microscopy have led to the development of alternative diagnostic methods such as polymerase-chain reaction (PCR)-based nucleic acid amplification tests that target specific genes or transcriptomes of the parasite at the erythrocytic stage. The commonly targeted genes or RNA transcripts and some recent advances in nucleic acid-based techniques have been extensively reviewed [15–18]. However, these alternative methods tend to be expensive, require trained personnel, lengthy turnaround time, and a level of sophistication that is not suitable for uptake in rural and poor healthcare settings [19]. The detection of a variety of other parasite-specific biomarkers including but not limited to histidine rich proteins 2 and 3 (HRP-2/3), lactate dehydrogenase (LDH), glutamate dehydrogenase (GDH), aldolase, merozoite surface protein 3 (MSP-3), and the biocrystal hemozoin have been explored order for a faster and easier diagnosis in the field. An extensive list of these biomarkers, as well as their metabolic role and clinical relevance, occurrence, genetic and structural organization, and kinetic parameters, have been discussed [1,20,21] and a brief overview is presented in subsequent sections of this review. Immunochromatographic tests have become popular for point-of-care (POC) diagnosis of malaria [13,22]. Commonly called a ‘rapid diagnostic test’ (RDT), it is based on a lateral flow immunoassay technique integrated into a cassette for single-step, cost-effective, simple and fast detection of parasite specific biomarkers. These attributes have made the RDTs immensely popular in the field for POC application since its introduction, with the distribution reaching a global estimate 1.92 billion RDT units between 2010 and 2017 [23]. Africa is the biggest consumer with more than 80% of the total RDT sales in 2017 alone (223 million out of the 276 million units) [2]. Plasmodium falciparum histidine rich protein 2 (PfHRP-2) is the main target for RDTs that detect P. falciparum infection. A variant of LDH, the pan-specific LDH (pLDH) and pan-specific aldolase common to all species are used in combination with the PfHRP-2 either for P. falciparum alone or for mixed infection [24]. Although RDTs have dominated point-of-care-tests (POCT) for malaria, there have been major concerns about the stability and performance relating to sensitivity and specificity which constrains their impact [25–27]. Currently, the commercially available RDTs are about 1000-fold lower in sensitivity than alternative laboratory-based techniques [28–30], thus they do not provide the sensitivity and quantitation comparable to the gold-standard microscopy or PCR [29]. Studies have reported that RDTs which incorporate pan-aldolase have poor sensitivity due to low expression of the enzyme by parasites. As a result, only few RDTs combine pan-aldolase/PfHRP-2 [31,32]. Similarly, poor sensitivity in pLDH-based RDTs are often associated with low parasitemia and high tropical temperature [33]. This gap and limitations necessitate the need to develop other diagnostic technologies with improved sensitivities [34,35], while ensuring simplicity, robustness, and cost-effectiveness. Some of these recent advances have included chip-based microfluidics, surface plasmon resonance and biosensors, many of which have achieved comparable sensitivities with traditional diagnostic methods. Sensors 2020, 20, 799 4 of 21 3. Biosensors for the Detection of Malaria Biomarkers in Clinical Samples Biosensors and immunosensors have experienced unprecedented growth in recent years and seem to be the most promising sensing tools with several analytical benefits and cost efficiency [36,37]. This growth has been driven in part by the surge in demand for POC devices in clinical diagnosis where biological sensing is integrated with microelectronics to form portable analytical devices. To date, nearly sixty years after the first biosensor for glucose detection, the technology has been widespread in several fields of analyte detection [38]. Glucometers have evolved enormously, receiving vast commercial success [39] whereas biosensors for other diseases have been limited to experimental research. Among the types of sensors, electrochemical biosensors have received considerable interest in clinical diagnostics owing to key advantages in their design, assay simplicity, and superior analytical performance over conventional laboratory methods [40,41]. These qualities make them suitable for POC application amidst efforts to improve and miniaturize electrochemical systems for portable devices. Most attempts to create miniaturized electrochemical devices for on-site analysis have applied screen-printed electrodes (SPE) as transducers and various nanomaterials as signal amplification strategies to improve the assay sensitivity [42–45]. Electrochemical immunosensors have been commonly applied to malaria diagnostic research given the benefits of low detection limits, wide linear response range, stability and reproducibility [46]. The strategies for detection comprise either a labelled assay in which apply amperometry and colorimetry or an impedimetric strategy, attractive for highly sensitive label-free detection [47,48]. Only a few potentiometric techniques have been reported [49]. The performances of selected biosensors reported for malaria biomarkers detection is summarized in Table 2. The choice of PfHRP-2 and LDH is still predominant, similar to RDTs. However, there is an increase in preference for pLDH possibly due to the persistence of PfHRP-2 antigenemia for several weeks after parasite clearance [50] and reports of mutant strains from Africa and Asia with deleted PfHRP-2 genes [51–53]. Sensors 2020, 20, 799 5 of 21 Table 1. Summary of selected biosensors reporting detection of various malaria biomarkers. Analytes Sensing Technique/ Response Transducer Biomarker Receptor Molecule LoD Range Response StorageTime Stability References 8.3–8.7 pM Colorimetric - pLDH (PvLDH,PfLDH) pL1 aptamer (PvLDH) 10.3–12.5 pM NA NA NA [54] (PfLDH) ** 108.5 fM EIS Gold electrode pLDH pL1 aptamer for PvLDH** 120.1 fM NA NA NA [55] for PfLDH EIS GCE pLDH P38 aptamer (90 mer ssDNA) 0.5 fM NA - NA [56] EIS GCE HRP-2 Anti-HRP-2 antibody ** 6.8 ag/mL. 10 ag/mL–10 mg/mL NA 2 months(86.5%) [57] Chemiresistive 15 days (electrical conductance) - PfHRP-2 Anti-HRP-2 antibody 0.97 fg/mL 10 fg/mL–10 ng/mL NA (94.2%) [44] - - PfHRP-2 Anti-PfHRP-2 0.025 ng/mL 0.01–10 ng/mL - - [58] EIS Gold discelectrodes Pf GDH ssDNA aptamer (NG3) * 0.77 pM 100 fM–100 nM NA NA [59]Antigens Potentiometric (FET) Gold Pf GDH ssDNA aptamer (NG3) ** 16.7 pMmicro-electrodes * 48.6 pM 100 fM–10 nM 5 s [49] Amperometric Gold-SPE PfHRP-2 Anti-PfHRP 2 mAb ** 36 pg/mL* 40 pg mL NA NA NA [60]/ Amperometric Gold-SPE pLDH pLDH capture antibody ** 19 pg/mL* 23 pg mL - - - [61]/ Spectrophotometric Indicator displacement medium - PfHRP-2 NA 30 ± 9.6 nM 10–100 nM 5 min NA [62] Colorimetric - PfLDH 2008s-biotin DNA aptamer ** 4.9 ng/mL NA <1h 2 months [63] Colorimetric - PfLDH 2008s aptamer - NA 20 min - [64] Amperometric SPE PfHRP-2 Mouse anti-PfHRP-2 antibody ** 8 ng/mL NA NA NA [65] FRET - pLDH Fluorescently-labeled aptamer(36 mer ssDNA) ** 550 pM NA NA NA [66] Amperometric magneto Immunosensor - PfHRP2 Anti-HRP2 IgM Antibody 0.36 ng/mL 0.35–7.8 ng/mL NA NA [67] Antibodies SPR Gold disc Antibodies of Pf. PfHRP2 ** 5.6 pgfor mAb - NA NA [68] Sensors 2020, 20, 799 6 of 21 Table 2. Summary of selected biosensors reporting detection of various malaria biomarkers. Analytes Sensing Technique/ Response Transducer Biomarker Receptor Molecule LoD Range Response StorageTime Stability References Quartz Crystal Microbalance - Pf msp2 gene Biotinylated probe ≥0.025 ng/mLof target DNA NA NA 180 days [69] Droplet Microfluidic Platform - Pf topoisomeraseI ds DNA substrate NA NA NA NA [70]Nucleic acids SERS Nanoplatform - Pf DNA Magnetic bead and nanorattle 100 attomoles 10−11 −10sequences –10 M NA NA [71] Quartz Crystal Microbalance Silver electrode 18s rRNA gene(Pf and Pv) immobilized probe - - [72] EIS SPE Pf infected RBCs monoclonal antibody - 102–107 cells/mL NA NA [73] Infected red blood cells microfluidic separation and MRR - Infected RBCs - 0.0005%parasitemia - - - [74] LoD: limit of detection; * LoD: LoD in real samples; ** LoD: LoD in buffer; EIS: electrochemical impedance spectroscopy; FET: field effect transistor; FRET: fluorescence resonance energy transfer; GCE: glassy carbon electrode; MRR: magnetic resonance relaxometry; SPE: screen-printed electrode; SERS: surface-enhanced Raman spectroscopy; SPR: surface plasmon resonance; SWV: square wave voltammetry. Sensors 2020, 20, 799 7 of 21 3.1. Detection of PfHRP-2 in Clinical Samples Histidine-rich protein 2 (HRP-2) is specific to P. falciparum (PfHRP-2) and is secreted into peripheral blood during parasite growth and development where it plays a role in heme detoxification. The antigen’s widespread application in electrochemical and optical immunosensors is due to copious expression levels throughout the parasite life cycle. Although primarily abundant in blood, trace amounts can be found in cerebrospinal fluid, urine, and saliva of infected patients [75,76], which offer an opportunity for non-invasive testing. Nonetheless, blood is preferred because of small sample volumes required to target the antigen. Painless testing has attractive public health benefits of voluntary testing and participation in screening programs geared towards malaria control [77]. However, only a few publications have attempted urine or saliva analysis as noninvasive malaria diagnostics [78]. Electrochemical techniques have been shown to outperform optical methods in many modelled POC tests. Nanoparticles, primarily gold (AuNP) have been adopted in signal amplification for amperometric immunosensors [79–81]. Their small size and ease of immobilizing bioconjugate probes allow for increased surface concentration of enzyme-tagged detection antibodies, hence higher signals from the catalytic reaction of enzyme and substrate. Sharma et al. were first to report an electrochemical immunosensor to detect PfHRP-2 in blood by amperometry [65]. The disposable immunosensor utilized multi-walled carbon nanotubes (MWCNTs) and gold nanoparticles (Nano-Au) to modify screen printed electrodes (SPE); resulting in Nano-Au/MWCNT/SPEs onto which rabbit-derived anti-PfHRP-2 were immobilized as capture antibodies. A sandwich enzyme-linked immunosorbent assay format was employed for the biosensor with alkaline phosphatase (ALP)-conjugated antibodies. Amperometric measurements were applied using ALP hydrolysis of 1-naphthyl phosphate. The Nano-Au/MWCNT/SPE had a limit of detection (LoD) of 8.0 ng/mL (compared to 80.0 ng/mL for bare SPE and 20.0 ng/mL for MWCNT/SPE) (Table 2). This enhanced performance was attributable to the synergistic effect of MWCNTs and AuNP. More importantly, the immunosensor had a superior analytical performance compared with a commercial immunochromatographic lateral flow test in the analysis of microscopy positive patient sample (sensitivity: 96% vs. 79%, specificity; 94% vs. 81% respectively). In assessing exposure to malaria parasites, recombinant PfHRP-2 was used as a recognition element for anti-PfHRP-2 antibodies in an amperometric immunosensor for early stages of malaria and at low parasitemia [82]. SPEs were modified with alumina sol-gel (Al2O3) and AuNP to obtain AuNP/Al2O3 sol-gel/SPE after which PfHRP-2 was bound. Rabbit anti-PfHRP-2 and anti-rabbit IgG-ALP conjugate were directed against capture antigens and the analytical responses determined by amperometry. In comparison to ‘gold standard’ microscopy, the immunosensor exhibited a sensitivity of 92% and a specificity of 90%. In another study that detected monoclonal antibodies to recombinant PfHRP-2 (MoaPfHRP-2), a gold chip was pre-treated with 4-mercaptobenzoic to immobilize recombinant PfHRP-2, then monitored for interactions between the antigen and antibody [68]. Label free surface plasmon resonance (SPR) screening of the interaction between the recombinant protein and target antibody produced a LoD of 5.6 pg (Table 2). Magnetic nanoparticles (MNPs) have also been applied in the development of a highly sensitive malaria immunosensor. Anti-HRP-2 was covalently attached to MNPs as capture elements and a second monoclonal antibody that binds a different epitope of the target antigen was labelled with horse radish peroxidase (HRP) to provide an electrochemical signal [67]. The anti-HRP-2-MNPs were captured onto a magnetic graphite-epoxy composite electrode incubated with HRP-2-spiked serum and anti-HRP-2-HRP in a sandwich assay format. Amperometric measurements produced an LoD of 0.36 ng/mL (Table 2), much lower than Sharma et al. [65] reported. Translating this strategy to the field would require magnetic supports for electrodes [67]. More recently, Hemben et al. used anti-PfHRP-2 monoclonal antibodies to capture PfHRP-2 at the surface of a screen printed gold electrode (Table 2) [60]. The captured antigen was targeted with HRP-labelled antibodies and the quantification of PfHRP-2 derived from the substrate (TMB-H2O2)-enzyme reaction by amperometry (Figure 2A). The LoDs in buffer and spiked human Sensors 2020, 20, 799 8 of 21 samples were determined as 2.14 ng/mL and 2.95 ng/mL, respectively. Labelled antibodies were subsequently conjugated to gold nanoparticles (AuNP) to amplify the sensor signal which improved Stehnesorsse 2n0s2i0t, i2v0i,t xy FaOnRd PLEEoRD RiEnVIbEuWff er (36.0 pg/mL) and spiked serum samples (40.0 pg/mL). 9 of 21 (A) (B) FFigiguurree 22.. SStrtarateteggyyf orfo(rA )(lAab) elllaebdeallmedp earommpeetrroicm[e6t0r]ica n[d60(B] ) alanbde l f(rBe)e ilmabpeeld ifmreeet riicm[p57e]deimlecettrroicc h[e5m7]i cal edleecttercotciohnemoicfalP dfHetRecPt-i2o.n o(f AP)fH(iR)PG-2o. l(dA)n (ain) oGpoaldrt inclaenoapmarptliicfilee damspanlifdiewdi cshanadswsaicyh aanssday( iai)ndp l(oiit) of pclhorto onfo cahmropneoraompeterriocmreestproicn rseesopfoPnfsHe RofP P-2fHdeRtPec-2ti odnetiencstipoink eind ssperiukemd (s0e.0ru5–m0 .(50n.0g5/–m0L.5 PnfgH/mRPL) P. RfHepRrPin).t ed RferopmrinHteedm frboemn eHt eaml. b[6e0n] ewt iathl. p[6e0r]m wisitshio pnefrrmomissMionD PfrIo.m(B M) (Di)PCIZ. (nBO) N(i)F CdZisnpOeNrsFio dnisspderorspio-cnass tderdopo-nto cGasCteEdf oolnlotow eGdCbEy fcohlleomwiesdo pbtiyo nchoefmanistoi-pHtiRonP 2ouf natnotiM-HPRAPt2r euantetdo eMlePcAtr otdreeastaendd e(lieic)tcroadlibesr aatinodn (ciui)r ve coaflibimraptieodni mcuertvreic orfe ismpopnedseims oetbrtiac irneesdpoanftseers ionbctuabinaetdin agftGerC iEn/cfuCbuaZtinnOg NGCFsE//AfCnutiZ-HnORPN2Fsb/iAosnetin-HsoRrPw2 ith bvioarsyeninsgorc ownictehn vtraartyioinngs ocof nPcfHenRtrPa2ti(o1n0sa ogf/m PLfH–1R0Pµ2g (/1m0 La)g. /(mAdLa–p10te µdgfr/momL)P. a(Auldeatpatle.d[5 f7r]owmi tPhapuelr met iassl.i on [5fr7o]m wiEthls epveiremr)i.ssion from Elsevier). 3.2. DWetehctilioenb oiof pseLnDsHin ign pCllaintfiocarlm Ssamfoprlems ost disease biomarkers tend to rely on antibodies as capture molePcluaslemso, daicuhma llleanctgaetew ditehhiymdmrougneonaasssea y(spiLsDreHla)t epdlatyosa nat icbaotdaylysttica brioliltey , ianp rtheree qgulyisciotely. tSico mpeatahtwteamyp ts dtourciinrgcu tmhev iennttrathereystehdroracywtibca sctkasghesa voef iPnlcalsumdoeddiugme. nIet tiisc pmraondiupcuelda tbioyn ms tehtaabt oimlicparlolyv eactthivees tpaabrialistiyteasn d wshitehlfin-l iifnefoecftaendt irbeodd bielosoadn dcetlhlse (uRsBeCosf)s [y8n6t–h8e8t]i canaldte hransa tciovnessesruvcehd acsataapltyatmic erress.idFuoerse xina malpl Plel,arsemsoedairucmhe rs scplopn. eedxcaenpdt ienx pPr. eksnsoewdlcesDi.N UAnlfirkaeg mHRenPt-s2e, npcLoDdHin gist hinedvicaartiiavbel eodf oam reacinenst( VinLf-eCctLioann adnVdH is- CgHen1e)roaflltyw o cmleoanreodc lowniathl iann t2i4b ohd ioefs pagaraainsistteP cflHeaRrPa-n2ce(F; 1h5e4n6caen, dit Fm11o1re0 )rienliEabsclhee rinic hidiaecnotliif[y8i3n]g. Trehceenretc uomnrbeisnoalvnetdF ab ifnrfaegcmtioennst.s showed similar binding properties to those of the parental monoclonal antibodies (mAb). ThisTahpeprero asecehmpsr otpoo sbees aa cgorsotw-eiffingci etnretnadlt eorfn aatpivtaemtoerl-abragsee-dsc asleenasnotrisb otadrygeptriondg upcLtiDonHf o[r54d,i5a5g,8n9o]s. tic Caopmplpicaarteido ntow aitnhtitbhoedoiepsp, oarptutanmiteyros faerne gsimneaelrleerd iann tsiibzoed, ythferramgmosetanbtslew, ietxhteimndperdo vsehdelaf ffilifnei twy,istthaobuilti ty, faunndctrioesniaslt adnecgertaodadteinonat, uarfafotirodnabevlee,n eawsiitlhy psyronltohnegsiezdedst oanradg ecainn uben croenatdriolyll emdotdemifipeedr. aIttu croesulidn, tthoe afine ld. extent be an alternative remedy in overcoming difficulties associated with using antibody-based tests. The unit costs for malaria RDTs in many African countries falls within the unsubsidized range of USD 2.54–2.83. Yet, a study in Uganda revealed that consumers were willing to pay an average of USD 0.53 [90]. The prospects of some biosensors/aptasensors being estimated at 1 USD or less per test, proposes a significant reduction to the general healthcare costs in impoverished tropical regions where malaria is prevalent. Sensors 2020, 20, 799 9 of 21 Besides using molecular labels and nanoparticles for improved diagnostics, some assays can probe the intimate recognition between the receptor and target alone. The benefits of using such label-free formats include a reduction in the assay complexity, preparation time, and analysis cost as it eliminates potentially confounding chemical labels. These strategies are better suited to field applications and under resourced settings where laboratories and skilled personnel are unavailable. A label-free a piezoelectric immunosensor for PfHRP-2 was designed by. applying mixed self-assembled monolayers (SAMs) of thioctic acid and 1-dodecanethiol on gold quartz crystal microbalance (QCM) Anti-PfHRP-2 antibodies were covalently immobilized unto the SAM-modified electrodes via EDC-NHS activation and the frequency change resulting from binding of different concentrations of PfHRP-2 measured [84]. The immunosensor exhibited a LoD of of 12.0 ng/mL with a linear range of 15.0–60.0 ng/mL for analysis of PfHRP-2 in buffer. This was higher than their previously reported amperometric immunosensor (8 ng/mL [65] vs. 12ng/mL [84]), and weaker responses were observed for PfHRP-2 concentrations lower than 25.0 ng/mL. However, application of the sensor to clinical samples produced comparable sensitivities with a commercial immunochromatographic test (ICT) kit (NOW® Malaria, Binax, Inc., Scarborough, Maine, USA). An indicator displacement assay (IDA) was used to detect and quantify HRP-2 in sera [62]. The label-free spectrophotometric method did not require any biorecognition elements and was based on the color change of murexide either in complex with nickel (Ni2+) or free in solution. In the IDA, competition between HRP-2 and Ni2+ displaces murexide from the murexide-Ni2+ complex. The resultant color intensity was proportional to free unbound murexide is measured to quantify HRP-2. The assay had LoD of 30.0 ± 9.6 nM (dynamic range of 10–100.0 nM) without any interfering signals from serum proteins (Table 2). Electrochemical impedance spectroscopy (EIS)-based methods present with numerous advantages that make them suitable candidates for POC application [85]. Their high sensitivity is evident from lower LoDs they tend to produce compared with other electrochemical methods. The lowest LoD yet reported for malaria was achieved by impedimetric detection of PfHRP-2 [57] (Table 2). In the sensor design, copper doped zinc oxide nanofibers (CZnONF) was drop-casted on glassy carbon electrode (GCE/CZnONF) followed by SAM modification and chemisorption of anti-PfHRP-2 (Figure 2B). The highly sensitive nanosensor (28.5 kΩ/(g/mL)/cm3) attained a detection limit of 6.8 ag/mL. The authors subsequently reported a flexible chemiresistive immunosensor in which the transducer comprised a 1-dimensional MWCNT-zinc oxide (MWCNTs-ZnO) nanofiber drop-casted on micro gold electrodes [44] (Table 2). Capture antibodies, anti-PfHRP-2 were immobilized on MWCNTs-ZnO by EDC-NHS crosslinking and resistance changes (∆R) measured to monitor the formation of PfHRP-2-anti-PfHRP-2 complexes. The device demonstrated good analytical performance as well as potential towards the development of POCTs with a linear response ranging from 10 fg/mL to10 ng/mL, LoD of 0.97 fg/mL and high specificity for PfHRP-2 over non-specific antigens. 3.2. Detection of pLDH in Clinical Samples Plasmodium lactate dehydrogenase (pLDH) plays a catalytic role in the glycolytic pathway during the intraerythrocytic stages of Plasmodium. It is produced by metabolically active parasites within infected red blood cells (RBCs) [86–88] and has conserved catalytic residues in all Plasmodium spp. except in P. knowlesi. Unlike HRP-2, pLDH is indicative of a recent infection and is generally cleared within 24 h of parasite clearance; hence, it more reliable in identifying recent unresolved infections. There seems to be a growing trend of aptamer-based sensors targeting pLDH [54,55,89]. Compared to antibodies, aptamers are smaller in size, thermostable, extended shelf life without functional degradation, affordable, easily synthesized and can be readily modified. It could, to an extent be an alternative remedy in overcoming difficulties associated with using antibody-based tests. The unit costs for malaria RDTs in many African countries falls within the unsubsidized range of USD 2.54–2.83. Yet, a study in Uganda revealed that consumers were willing to pay an average of USD 0.53 [90]. The prospects of some biosensors/aptasensors being estimated at 1 USD or less per test, proposes a Sensors 2020, 20, x FOR PEER REVIEW 10 of 21 In a prospective tool for asymptomatic and early diagnosis of malaria, single-stranded DNA aptamers (pL1 aptamers) were used to target recombinant Plasmodium falciparum LDH (PfLDH) and Plasmodium vivax LDH (PvLDH) in buffer and in real samples [55] (Table 1). Impedance measurements for the interaction between pL1 and the target proteins demonstrated high sensitivity and specificity with LoDs measuring 108.5 fM (for PvLDH) and 120.1 fM (PfLDH). Native pLDH in clinical samples was also detected up to 1 parasite/µL. Figueroa-Miranda et al. immobilized 2008s aptamers on a SAM-modified gold electrode to bind pLDH down to the detection limit of 0.84 pM in buffer and 1.30 pM in blood (Figure 3, Table 1) [89]. The aptasensor had a detection range between 1 pM–10 nM and remained highly selective for PfLDH even in the presence of high concentrations of serum proteins and analogues of LDH from muse muscle. The thiol-gold covalent bonding from SAMs conferred high immobilization stability to the aptamer and could be regenerated and re-used for up to three times without loss of analytical performance. A major observation was the influence of isoelectric point (pI) of PfLDH on impedimetric responses which tended to increase where pH > pI and a decrease at pH < pI. A likely reason for this occurrence is attributable to a repulsion and attraction of the redox probe to the electrode surface. SensorAs 2p0t2a0m, 2e0,r7 9f9unctionalized microbeads were used to determine the capture and measur1e0 othf 2e1 intrinsic enzymatic activity of LDH in a colorimetric assay (Figure 4A) [63]. In the aptamer-tethered esnigznyimficea cnatprteudruec t(iAoPnTtoECth) eagsesanye,r atlhhe ebaeltahdcsa rceocnofsetrsreind iam pwoidveer issuhrefadcter oapriecaa lforerg aionnaslywteh ebrinedminagla rtioa pisropdreuvcae laen Lt.oD of 4.9 ng/mL for recombinant PfLDH (Table 1). Further work integrated the APTEC assayI ninatop aro pspoertcatbivlee tmooiclrfooflruaidsyicm bpitoosmenastoicr a(Fnidgueraer ly4Bd) ia[6g4n]o. sTisheo fpmlaatfloarrmia, rseinsogllvee-sdt rsaonmdeed oDf NthAe aaspstaaym’se irnsit(ipaLl l1imapittaatmioenrss )ofw laerregeu ssaemd ptolet aarngde treraegcoemntb vionlaunmt Pesla wsmhoildei udmetefacltciinpga rPu.m falLcDipHaru(Pmf LwDitHh )haignhd sPpleascmifiocdiituym anvdiv asxenLsDitHivi(tPyv iLnD cHul)tuinrebsu affnedr acnlidniicnalr esaalmspamleps.l es [55] (Table 2). Impedance measurements for thFeluionrteesrcaecntitolyn-blaebtwelleeedn appLt1amanedrst wheertae ragdestoprrboetdei ntos mdeomlyobndsiturmate ddishuilgphhsidene s(iMtivoiSty2) annadnosspheeceifitsc ittoy dweivtheloLpo Da sFRmEeTas auprtiansgen10so8.r5 wfMhic(hfo srePlevcLtiDveHly) adnedtec1t2e0d.1 pfLMDH(P fiLnD a Hhe).teNroagtievneepoLuDs pHrointecinli nmicixaltusraem [6p6le].s Twhaes malesochdaentiescmte dofu tpheto a1sspaayr awsiatse /bµaLs.ed on a “capture-release” model whereby fluorescence of the aptamFeigr uise rqouae-nMcihreadn duapeotna al.pitma meorb-MiliozeSd2 n2a0n0o8sshaepettasm beinrsdoinnga aSnAd Mre-smtoordedifi iend tghoel pdreelseecntrcoed oef tpoLbDinHd wpLhDenH thdeo wapntatomtehre isd erteelecatisoend lifmroimt o tfh0e. 8n4apnMoshineebtus.ff Tehr ea nadtta1c.3h0mpeMnt iannbdl odoedta(cFhigmuernet 3p, rToacbelses2es) [a8r9e] . fTahcieliataptteads ebnys otrheh ahdigahd aeftfeinctiitoyn breatnwgeeebne atwpteaemne1rsp Man–d1 0pLnDMHa.n Tdhree mseanisnoerd ahcihgihelvyesde lLeocDtiv oeff 5o5r0P.0fL pDMH (eTvaebnlein 1)th. e presence of high concentrations of serum proteins and analogues of LDH from muse muscle. The tHheioml-bgeonld etc oalv. afluenncttbioonnadliiznegdf rsocrmeeSnA-pMrisntceodn fgeorlrde delheicgtrhoidmesm (oSbPiGliEza) twioinths atanbtii-liptyLDtoHt haentaipbtoadmieesr aanndd acpopullidedb ae sreagnednweircaht eadssaanyd forerm-uaste tdo fdoertuecpt tpoLtDhHre e[6t1im]. eTshwe sitehnosuotr lionsitsiaolflya nacahlyietivceadl pLeorDfosr omf a1n.8c0e . nAg/maLjo irno bbusfefrevr aatniodn 0w.7a0s ntgh/eminLfl inu esnecruemof. iAsopeplelicctartiicopno oifn cto(pllIo)idoaf lP AfLuDNHPso fnuinmctpioendaimlizeetdri cwrietshp HonRsPe-s lwabheilclhedte dnedteedcttioonin acnretiabsoedwiehse erenhpaHnc>edp Iaamnpdearodmecerteraics esiagtnpaHls