Biosensors for the detection of flaviviruses: A review

Re iew A icle Biosenso s o he de ec ion o la i i uses: A e iew Ana-Bel´ en Bl´ azquez , Ne eida Jim´ enez de Oya * Depa amen o de Bio ecnología, Ins i u o Nacional de In es igaci´ on y Tecnología Ag a ia y Alimen a ia (INIA-CSIC), C a. de La Co u˜ na, km 7.5, 28040 Mad id, Spain ARTICLE INFO Keywo ds: Fla i i uses Biosenso s Poin -o -ca e es ing (POCT) Diagnos ic ABSTRACT Fla i i uses a ec he li es o millions o people in endemic egions and also ha e he po en ial o impac non- endemic a eas. Fac o s such as clima e change, global wa ming, de o es a ion, and inc eased a el and ade a e linked o he sp ead o la i i uses in o new habi a s and hos species. Gi en he absence o speci ic ea men s and he limi ed a ailabili y o accines, i is impe a i e o unde s and he biology o la i i uses and de elop apid and sensi i e diagnos ic es s. These measu es a e essen ial o p e en ing he ansmission o hese po en ially li e- h ea ening pa hogens. Fla i i us in ec ions a e mainly diagnosed using con en ional me hods. Howe e , hese echniques p esen se e al d awbacks, including high expenses, ime-consuming p ocedu es, and he need o skilled p o essionals. The sea ch o as , easy- o-use, and a o dable al e na i e echniques as a easible solu ion o de eloping coun ies is leading o he sea ch o new me hods in he diagnosis o la i i- uses, such as biosenso s. This e iew p o ides a comp ehensi e o e iew o di e en biosenso de ec ion s a egies o la i i uses and desc ibes ecen ad ances in diagnos ic echnologies. Finally, we explo e hei u u e p ospec s and po en ial applica ions in pa hogen de ec ion. This e iew se es as a aluable esou ce o unde s and ad ances in ongoing esea ch in o new biosenso -based diagnos ic me hods o la i i uses. 1. In oduc ion The genus Fla i i us, ecen ly enamed O ho la i i us, is cons i u ed by a h opod-bo ne posi i e-sense single-s anded RNA i uses belonging o he amily Fla i i idae [1]. This genus comp ises mo e han 70 di e en species, classi ied in o 3 ypes acco ding o he ansmission ec o : mosqui o-bo ne, ick-bo ne, and unknown- ec o -bo ne i uses. Fla i i uses include some o he mos impo an human pa hogens such as dengue i us (DENV), yellow e e i us (YFV), Japanese encephali is i us (JEV), Zika i us (ZIKV), Wes Nile i us (WNV), and ick-bo ne encephali is i us (TBEV), causing a majo global heal h conce n [2]. Fla i i us in ec ions display a wide a ie y o symp oms anging om asymp oma ic o mild e e o se e e mani es a ions, which could be di ided in o wo di e en ca ego ies: hemo hagic and neu ological complica ions [3]. The main hemo hagic ea u es o he disease can be li e ailu e, hemo hagic synd omes, and ascula comp omise, and may be a al. Neu o opic la i i uses can each he b ain and spinal co d and cause se e e neu ological synd omes such as meningi is, en- cephali is, and acu e laccid pa alysis [4]. On he o he hand, ZIKV in ec ion du ing p egnancy can be ansmi ed o he de eloping e us, esul ing in placen al insu iciency, mic ocephaly, congeni al mal o - ma ions, and e al demise. No speci ic an i- la i i al ea men s a e cu en ly a ailable, and only a ew accines ha e been app o ed o humans agains JEV, DENV, YFV, and TBEV, o ho ses in he case o WNV and JEV [5,6] and o pigs in he case o JEV [7]. The e o e, knowledge o he biology o la i i uses and he de elopmen o apid and sensi i e diagnos ic es s is c ucial o p e en he sp ead o hese po en ially li e- h ea ening pa hogens [4]. 1.1. Vi ological ea u es o la i i uses Fla i i uses a e en eloped RNA i uses. Thei genome is o med by a single-s anded posi i e RNA o app oxima ely 11 kb in size ha en- codes a polyp o ein wi hin a single open eading ame (ORF), lanked by un ansla ed egions (UTRs) a bo h he 5ʹ and 3ʹ ends. The ORF is ansla ed in o a single polyp o ein, which is p ocessed by i al and cellula p o eases o p oduce en majo i al p o eins: h ee s uc u al (C, p M/M, and E) and se en non-s uc u al p o eins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) [8] (Fig. 1). P o eins a e implica ed in di e en s eps o he eplica ion cycle. The Pee e iew unde esponsibili y o KeAi Communica ions Co., L d. * Co esponding au ho . E-mail add ess: [email p o ec ed] (N. Jim´ enez de Oya). Con en s lis s a ailable a ScienceDi ec Syn he ic and Sys ems Bio echnology jou nal homepage: www.keaipublishing.com/en/jou nals/syn he ic-and-sys ems-bio echnology h ps://doi.o g/10.1016/j.synbio.2024.10.005 Recei ed 27 July 2024; Recei ed in e ised o m 26 Sep embe 2024; Accep ed 21 Oc obe 2024 Syn he ic and Sys ems Bio echnology 10 (2025) 194–206 A ailable online 26 Oc obe 2024 2405-805X/© 2024 The Au ho s. Publishing se ices by Else ie B.V. on behal o KeAi Communica ions Co. L d. This is an open access a icle unde he CC BY-NC-ND license ( h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/ ). C p o ein is in ol ed in nucleocapsid o ma ion, an essen ial s ep o i al assembly and eplica ion [9]. The M p o ein is a ansmemb ane glycosyla ed p o ein esul ing om he clea age o he p M p o ein by a u in-like p o ease, leading o he o ma ion o ma u e i ions. The E p o ein is also a ansmemb ane glycosyla ed p o ein in ol ed in di e en p ocesses such as ecep o binding, i al en y, and memb ane usion. This p o ein is conside ed he mos immunogenic, and i s glycosyla ion is c ucial o e icien ansmission and neu oin asi eness. The NS1 is in ol ed in eplica ion, immunomodula ion, and pa ho- genesis. The NS2A pa icipa es in in acellula memb ane ea ange- men s and i ion assembly. The NS2B p o ein is he co- ac o o he NS3 i al se ine p o ease, allowing i s ac i a ion and he consequen p o- cessing o he i al polyp o ein. In addi ion, i in e ac s wi h he NS2A p o ein, playing a c ucial ole in i al eplica ion and assembly [10]. The NS3 is a mul i unc ional p o ein, wi h a cen al ole in in ec i i y, allowing he ma u a ion o i al p o eins h ough i s p o ease ac i i y, bu i also p esen s helicase, nucleoside iphospha ase, and RNA i- phospha ase ac i i ies in ol ed in i us eplica ion [11]. The NS4A is in ol ed in memb ane ea angemen s, inhibi ion o IFN signaling, and is ela ed o impo an p ocesses such as au ophagy o un olded p o ein esponse. The NS4B pa icipa es in he o ma ion o he i al eplica ion complex. The NS5 is he mos conse ed p o ein among he di e en la i i uses. I s me hyl ans e ase enzyma ic ac i i y is necessa y o he i al RNA capping and i s RNA-dependen RNA polyme ase (RdRp) ac i i y o he eplica ion o he i us genome [12]. Replica ion s a s wi h i al en y in hos cells by ecep o -media ed endocy osis. Vi ions bind o ecep o hos endosomes in an acidic en i onmen , allowing he usion o he i al en elope wi h he endo- somal hos memb ane. Then he i al genome is eleased in o he cy osol [13]. In ec ious i ions eme ged when imma u e i al pa icles assem- bled a he endoplasmic e iculum each he Golgi complex o ma u- a ion. A e his p ocess, i al pa icles a e eleased om he in ec ed cell o he ex acellula space by exocy osis (Fig. 2). 1.2. Geog aphic dis ibu ion and clinical mani es a ions The wo ldwide geog aphic dis ibu ion o la i i uses is well known. E en hough hese i uses a e de ec ed mainly in opical and sub op- ical a eas, ac o s such as clima e change and global wa ming, de o - es a ion, uncon olled u baniza ion o a eling and ade a e associa ed wi h la i i uses colonizing new habi a s and hos species [14] hus con ibu ing o he inc ease o la i i al in ec ions in o p e iously non-endemic a eas. Dengue i us (DENV) causes mo e han 90 million cases and app oxima ely 40000 dea hs annually [15], being he mos widesp ead a bo i us. Acco ding o he Wo ld Heal h O ganiza ion (WHO), DENV cases ha e been epo ed in o e 80 e i o ies in A ica, he Ame icas, Sou heas Asia, he Wes e n Paci ic, and Eas e n Medi e anean Regions du ing 2023. Pa icula ly wo ying is he ac ha almos 80 % o hese cases occu ed in he Ame icas [16], whe e cyclic epidemics ecu ing e e y 3–5 yea s ha e been epo ed. Mo eo e , au och honous dengue cases ha e also been desc ibed in he Eu opean egion, since i s mos- qui o ec o s a e inc easing hei p esence no hwa ds and wes wa ds in Eu ope [17]. Howe e , i is suspec ed ha he numbe o cases is unde es ima ed because mos o he in ec ions a e usually asymp oma ic. DENV in ec ions p o oke a mild disease called dengue e e which displays a di e se a ay o symp oms, such as e e , headache, and myalgia, which equen ly o e lap wi h hose o o he eb ile illnesses, posing a challenge o accu a e di e en ia ion wi hou app op ia e diagnos ic me hods. Howe e , in some cases, DENV can igge a mo e se e e disease known as dengue hemo hagic e e (DHF) o dengue shock synd ome (DSS), which p esen s po en ially li e- h ea ening symp oms like hemo hage, h ombocy openia, and ascula leakage [18]. Zika i us (ZIKV) was i s iden i ied in 1947 in Uganda, and i emained in he A ican con inen un il i s de ec ion in Sou heas Asia in he 1980s, hen in Mic onesia and Oceania beginning in 2007, and inally, he i us eached he Ame icas in 2015 whe e i p o oked an explosi e ou b eak, in ec ing hund eds o housands o people, mainly a ec ing p egnan women and newbo ns [19]. E en hough ZIKV dis- ease is usually asymp oma ic o p esen s mild symp oms, se e e neu ological mani es a ions such as Guillain-Ba ´ e synd ome (GBS) and mic ocephaly in newbo ns ha e been widely epo ed in he Ame icas [20]. Wes Nile i us (WNV) is cu en ly conside ed one o he mos impo an causa i e agen s o human i al encephali is wo ldwide [21]. The i us was i s epo ed in he Wes Nile dis ic o Uganda in 1937 [22]. In he ollowing decades, WNV was conside ed a neglec ed Fig. 1. Schema ic iew o he genomic o ganiza ion o genus O ho la i i us. UTR: un ansla ed egion; C: capsid o co e p o ein; p M: p e-memb ane p o ein; E: en elope p o ein; NS: non-s uc u al p o eins. Fig. 2. Schema ic iew o la i i uses in ec ious cycle. The majo s eps o in ec ion, including ecep o -media ed endocy osis, genome eplica ion, imma u e i ion in he endoplasmic e iculum, pa icle ma u a ion, and ma u e i ion elease by exocy osis a e schema ized. A.-B. Bl´ azquez and N. Jim´ enez de Oya Syn he ic and Sys ems Bio echnology 10 (2025) 194–206 195 pa hogen wi h in ec ions spo adically epo ed in A ica, Is ael, he Medi e anean Basin, Russia, and Aus alia [23]. I was no un il 1999 ha a WNV ou b eak occu ed in New Yo k ha sp ead explosi ely h oughou he Uni ed S a es in he ollowing yea s. Nowadays he i us is commonly ound in A ica, Eu ope, he Middle Eas , No h Ame ica, and Wes Asia. WNV is classi ied in o se e al lineages ha do no consis en ly co ela e wi h i s geog aphical dis ibu ion. Only lineages 1 and 2 ha e been in ol ed in human ou b eaks o WNV encephali is, and bo h a e now endemic in Eu ope. In ec ions a e mainly asymp oma ic o cause mild symp oms, bu a small pe cen age o in ec ed people (less han 1 %) de elop se e e neu oin asi e mani es a ions such as en- cephali is and meningi is ha can p oduce a al consequences [9]. Japanese encephali is i us (JEV) is he leading cause o i al en- cephali is in Asia, causing a ound 60000 cases e e y yea , and a 30 % mo ali y a e in hose wi h encephali is [24]. The disease is mainly de eloped du ing childhood, being endemic in 24 coun ies in Sou heas Asia and Wes e n Paci ic egions. Ou b eaks a e unp edic ably and spa ially and empo ally limi ed. Incidence in Asia has dec eased, mainly a ibu ed o accina ion [25]. Yellow e e i us (YFV) is ound in opical and sub opical a eas o A ica and Cen al and Sou h Ame ica. E en hough mos in ec ed people ha e no symp oms o mild ones, a small pe cen age o pa ien s can su e se e e complica ions in he li e and kidneys. They can de elop jaundice and abdominal pain wi h omi ing o bleeding. The e is a mo ali y a e o 50 % in hose pa ien s who en e he oxic phase [26]. Tick-bo ne encephali is i us (TBEV) is he main causa i e agen o a bo i al encephali is in Eu ope. The i us is endemic in his con inen and in egions o China and No h Japan in Asia [27]. Neu ological complica ions o he disease usually p esen as meningi is, meningoen- cephali is, o meningoencephalomyeli is. Be ween 10,000 and 12,000 clinical cases o ick-bo ne encephali is a e epo ed annually, bu he o al numbe o clinical cases is belie ed o be unde es ima ed [28]. 1.3. Diagnos ic me hods o la i i us iden i ica ion As p e iously men ioned, la i i uses a e globally dis ibu ed and p oduce dange ous li e- h ea ening in ec ions in opical and sub opi- cal a eas. In his con ex , he sea ch o apid and e icien diagnos ic me hods ep esen s a miles one in he con ol o la i i al diseases. Diagnosis o la i i us in ec ions is gene ally achie ed by con en- ional me hods, including molecula and se ological assays (Table 1). In his sense, he mos used molecula me hods a e e e se ansc ip ion polyme ase chain eac ion (RT-PCR) and eal- ime quan i a i e RT-PCR. The main disad an age is hei high cos , which makes hem una o d- able in low-income coun ies. On he o he hand, he window o i us de ec ion using hese echniques is ela i ely na ow, since hey mus be applied du ing he ea ly s age o he disease [29]. Among se ological me hods, he mos widely used a e enzyme-linked immunoso ben assays (ELISAs), used ei he du ing he acu e phase o de ec ea ly IgM an ibodies o in he la e phases o in ec ion o de ec IgG an ibodies. Howe e , i is essen ial o ake in o accoun ha c oss- eac i i y is e y common among la i i uses, educing he speci ici y o he diagnosis and p oducing alse posi i e esul s [30]. In his sense, he plaque educ ion neu aliza ion es (PRNT) is conside ed he gold s anda d echnique o he di e en ial se ological diagnosis o la i i us [23]. PRNT is e y speci ic and minimal c oss- eac i i y is obse ed be ween di e en la i i uses. This me hod is used o de ec neu alizing an ibodies. Howe e , he main d awback when wo king wi h in ec ious i uses is ha mos o hem can only be handled wi hin a biosa e y le el 3 (BSL-3) acili y. Ano he classical diagnos ic me hod is cell cul u e o i al isola ion. Howe e , and as happened wi h PRNTs, hese p ocedu es may be con- duc ed a designa ed esea ch acili ies placed in BSL-3 labo a o ies. The main d awbacks o he applica ion o classical diagnos ic me hods a e he high economic and quali ied pe sonnel equi emen s, in addi ion o he ac ha hey a e ime-consuming me hods and equi e expensi e echnical equipmen and labo a o y acili ies. The sea ch o as , simple, and a o dable al e na i e echniques as a easible solu ion o de eloping coun ies is leading o he explo a ion o new me hods o he diagnosis o la i i uses, such as biosenso s. 1.4. Biosenso s 1.4.1. Gene al p inciples Biosenso s a e analy ical ools ha use chemical o biological mechanisms o iden i y pa icula a ge subs ances, p incipally including wo p ima y elemen s: a ecep o and a ansduce [31]. The bio ecep o a aches o he a ge analy e, iden i ied h ough physical o chemical in e ac ions. The ansduce con e s his eac ion in o a measu able signal. T ansduce s p oduce a wide a ie y o signals, usu- ally elec ochemical, op ical, acous ic, o calo ime ic. This signal ob- ained om he ansduce is usually ampli ied and analyzed by a de ec o (Fig. 3). The i s de ice conside ed a biosenso was de eloped by Leland C. Cla k, J in 1956 who designed an elec ode o oxygen de ec ion [32]. In 1962, Cla k and Lyons success ully de eloped an enzyma ic elec ode capable o con e ing glucose in o a de ec able signal, ma king he beginning o biosenso s as essen ial bioanaly ical ins umen s [33]. Biosenso s o e g ea po en ial such as ou s anding pe o mance, easy handling, high sensi i i y and speci ici y, and he abili y o p o ide a apid esponse and pe o m analysis in eal- ime, hus allowing apid in e en ion in case o heal h eme gencies [34]. In addi ion o hese ea u es, hei compac size and po abili y make hese de ices an ideal ool o poin -o -ca e es ing (POCT) in bioanaly ical clinics. Nowadays he e is a g owing need o POCT o he apid de ec ion o in ec ious diseases, such as hose caused by i uses. These de ices play an essen ial ole in p e en ing he sp ead o in ec ious diseases by enabling eal- ime es ing and p o iding apid, high-quali y diagnosis [35]. The Wo ld Heal h O ganiza ion (WHO) has highligh ed he ele- ance o de eloping POCT ha mee s he ASSURED c i e ia: A o dable, Sensi i e, Speci ic, Use - iendly, Robus and apid, Equipmen - ee, and Deli e able. These c i e ia ep esen he essen ial a ibu es o an op imal POCT pla o m [36] (Fig. 4). 1.4.2. Cha ac e is ics o biosenso s Biosenso s e ec i eness is de e mined by some essen ial ea u es: selec i i y, ep oducibili y, s abili y, limi o de ec ion, linea de ec ion ange, and esponse ime [32]. Selec i i y e e s o he capabili y o he bio ecep o o iden i y a de e mined analy e wi hin a sample ha may con ain a mix u e o compounds. I is p obably he mos impo an cha ac e is ic o biosenso s. Rep oducibili y indica es he capaci y o he biosenso o p oduce analogue esponses when expe imen al condi ions a e eplica ed. This ea u e is mainly de e mined by he p ecision and accu acy o he ansduce and he elec onics o he biosenso . The s abili y is a ibu ed o he suscep ibili y o he biosenso sys em o ex e nal dis u bances p esen in i s su ounding en i onmen , which induce luc ua ions in he ou pu signals o he biosenso du ing mea- su emen . In applica ions in ol ing long incuba ion pe iods o con in- uous moni o ing, s abili y becomes a c i ical cha ac e is ic. S abili y can Table 1 Classical me hods o he diagnosis o la i i uses. Molecula assays Se ological assays Vi al assays Re e se ansc ip ion polyme ase chain eac ion (RT-PCR) Enzyme-linked immunoso ben assay (ELISA) Plaque educ ion neu aliza ion es (PRNT) Real- ime quan i a i e RT- PCR (qRT-PCR) Cell cul u e o i al isola ion A.-B. Bl´ azquez and N. Jim´ enez de Oya Syn he ic and Sys ems Bio echnology 10 (2025) 194–206 196 also be in luenced by bio ecep o deg ada ion o e ime o when em- pe a u e changes occu in ansduce s o de ec o s. The limi o de ec ion (LOD) o a biosenso is conside ed he mini- mum amoun o analy e de ec ed by he de ice. This pa ame e is di ec ly ela ed o sensi i i y, which is de e mined by he co ela ion be ween he a ia ion in he concen a ion o he analy e and he in- ensi y o he signal moni o ed by he ansduce . Ideally, biosenso s would be able o p oduce a signal in esponse o e en sligh changes in he concen a ion o he a ge molecule [37]. Linea i y is he cha ac- e is ic ha shows he accu acy o he measu ed esponse o ollow a linea end when a ious analy e concen a ions a e de e mined. This linea de ec ion ange is also associa ed wi h he sensi i i y o he biosenso . Response ime is de ined as he equi ed ime o he biosenso o p oduce a signal o esponse a e in e ac ion be ween he ecep o and he a ge sample. I is gene ally aken as he ime needed o achie e 95 % o he esponse [38]. 1.4.3. Classi ica ion o biosenso s Di e en c i e ia a e used in he classi ica ion o biosenso s. The mos equen a e shown in Table 2, acco ding o he bio ecep o o ansduce chosen. This choice depends mainly on he cha ac e is ics o he a ge analy e and he ype o physical o chemical p ope y o be measu ed [39]. 1.4.3.1. Biosenso s based on bio ecep o s. As men ioned abo e, a bio- ecep o is a biomolecule ha uses a biochemical mechanism o iden i y an analy e. I s unc ion is o cap u e he analy e o in e es and a ach i o he senso o u he s udy. Bio ecep o s can gene ally be classi ied in o di e en ca ego ies including enzymes, an ibodies, nucleic acids, cellula s uc u es/cells, and o he mic oo ganisms. Enzymes and an ibodies a e he main ypes o bio ecep o s used in biosenso applica ions (Fig. 5). 1.4.3.1.1. Enzyme-based biosenso s. Enzyme-based biosenso s s and ou as one o he mos ad anced bioanaly ical ools, due o he high ca aly ic ac i i y and selec i i y o enzymes o de ec a ge analy es [38]. Thanks o he ex ensi e de elopmen o enzyme-based ecep o s, a wide a ie y o biosenso s can be gene a ed based on enzyme speci ici y. Ne e heless, con en ional enzyme-based biosenso s o en ace chal- lenges ela ed o hei sensi i i y, selec i i y, and s abili y. Conse- quen ly, di e en s a egies a e being explo ed o imp o e he pe o mance o hese biosenso s, including he in eg a ion o nanoscale ma e ials ha imp o e physical and chemical p ope ies [40]. 1.4.3.1.2. An ibody-based biosenso s. An ibody-based biosenso s o immunosenso s a e one o he mos impo an classes o a ini y bio- senso s due o hei speci ici y. These de ices con ain an embedded an ibody as a ligand. A speci ic a ge analy e, he an igen, o ms a s able immune complex wi h an an ibody ha ac s as a cap u e agen based on he an ibody-an igen in e ac ion [41]. This in e ac ion leads o he gene a ion o a measu able signal p o ided by a ansduce . Immuno- senso s ha e demons a ed ema kable selec i i y and sensi i i y due o p ecise an igen-an ibody binding, making hem highly sui able o a ious clinical applica ions, including pa hogen de ec ion [42]. 1.4.3.1.3. Nucleic acid-based biosenso s. The mos common bio- senso s ha use nucleic acids consis o single-s anded DNA, which hyb idizes wi h i s complemen a y s and, exhibi ing ema kable e i- ciency and speci ici y [43]. DNA senso s, also called genosenso s, a e an in e es ing ool o p o ide access o sequence-speci ic in o ma ion. This capabili y can be widely used ac oss nume ous ields, pa icula ly in clinical, en i onmen al, and ood analysis [44]. O he commonly used nucleic acid biosenso s ha e been gene a ed using ap ame s o mic oRNA [45]. An ap ame is a sho single-s anded nucleic acid, whe he ssDNA o RNA, ha binds o a speci ic a ge molecule [46]. Due o hei syn he ic and chemical simplici y unlike an ibodies, ap ame -based biosenso s o ap asenso s o e imp o ed s abili y and unc ionali y o de ec ing en i onmen al con aminan s [47] o o biomedical applica ions [48] among o he s. 1.4.3.1.4. Cell- and o ganelle-based biosenso s. Bio ecogni ion in cell-based biosenso s elies on he whole cell o on a pa icula cellula componen o o ganelle ha is compe en o speci ic binding o ce ain species [39]. Cell-based biosenso s in eg a e li ing cells wi h senso s o ansduce s o de ec cellula physiological pa ame e s, hus ac ing as a Fig. 3. Schema ic design o a biosenso . The main elemen s o a biosenso a e included: analy e, ecep o , ansduce , and de ec o . Fig. 4. Rep esen a ion o he assu ed c i e ia highligh ed by he Wo ld Heal h O ganiza ion (WHO). Table 2 Classi ica ion o biosenso s. Based on bio ecep o s Based on ansduce s Enzyme-based biosenso s Elec ochemical biosenso s An ibody-based biosenso s Op ical biosenso s Nucleic acid-based biosenso s The mal biosenso s Cell- and o ganelle-based biosenso s G a ime ic biosenso s Mic obial-based biosenso s Magne ic biosenso s A.-B. Bl´ azquez and N. Jim´ enez de Oya Syn he ic and Sys ems Bio echnology 10 (2025) 194–206 197 connec ion be ween biology and elec onics. These biosenso s p esen e iden ad an ages, such as p olonged non-in asi e eco ding, as esponse imes, and label- ee expe imen a ion [49]. In his sense, hey a e being used in a as a ie y o applica ions ha include he de ec ion o biologically ac i e signaling molecules, an imic obial s a egies, o cance he apy, among o he s [50]. Howe e , despi e hese ad an ages, he e a e s ill some obs acles, such as egene a ion and s o age li espan, cell popula ion he e ogenei y, signi ican in e e ence, and high cos s, which need o be esol ed be o e la ge -scale implemen a ion o cell-based biosenso s [51]. 1.4.3.1.5. Mic obial-based biosenso s. A mic obial-based biosenso is an analy ical ool p oduced by combining immobilized iable o non- iable mic oo ganisms wi h a physical ansduce o p oduce a measu able signal p opo ional o he concen a ion o he analy e [52]. The immobiliza ion o mic oo ganisms on ansduce s plays an essen ial ole in mic obial biosenso s, so he e is a huge a ie y o me hods o his immobiliza ion such as adso p ion, encapsula ion, co alen binding, e c. [53]. Al hough me aboli es p oduced by mic oo ganisms a e gene ally non-speci ic, achie ing highly selec i e mic obial biosenso s is po en- ially easible by excluding unwan ed me abolic pa hways and inducing ele an ones. This can be accomplished by adjus ing he mic oo gan- isms o sui able subs a es o in e es . Addi ionally, ecen ad ances in molecula biology ha e in oduced a no el app oach o c ea ing gene ically modi ied mic oo ganisms, o e ing a new way o imp o e he selec i i y and sensi i i y o mic obial biosenso s [54]. 1.4.3.2. Biosenso s based on ansduce s. As men ioned abo e, he ansduce essen ially wo ks as an in e p e e , de ec ing he in e ac ion o a ious biochemical eac ions and con e ing i in o ano he signal eady o be analyzed by he de ec o . Depending on he mechanism by which he ansduce s pe o m he con e sion, he signal gene a ed by he in e ac ion be ween bio ecep o and analy e can be di e en and, as shown in Fig. 6, biosenso s can be classi ied acco ding o he ans- duc ion me hods hey employ. 1.4.3.2.1. Elec ochemical biosenso s. T ansduce s depending on elec ochemical de ec ion mechanisms a e he mos commonly used in he de elopmen o biosenso s. Elec ochemical biosenso s ely on he in e ac ions be ween he bio ecogni ion elemen ha is included on i s su ace and he binding molecule p esen in he analy e. These in e ac ions induce changes in elec ochemical p ope ies, which subsequen ly ansla e in o a de ec - able elec ical signal. Elec ochemical biosenso s can be classi ied in o ampe ome ic, po en iome ic, impedime ic, conduc ome ic, ol am- me ic, pola og aphic, capaci i e, o piezoelec ic, depending on he de ec ion p inciple and applica ion [55]. On he o he hand, label- ee biosenso s cons i u e a ca ego y o elec ochemical biosenso s in which he quan i ica ion o he a ge analy es is based on he echniques desc ibed abo e bu no o he signal labels a e equi ed. The inclusion o a ag can modi y he speci ic binding o he analy e, leading o po en ial sys ema ic e o s in he measu emen . Di ec de ec ion elimina es he labeling s eps, educing he ime and cos o analysis [56]. Elec ochemical me hods o e signi ican ad an ages, including high sensi i i y, apid signal gene a ion and de ec ion, minia u iza ion, Fig. 5. Classi ica ion o biosenso s acco ding o he ype o bio ecep o . Fig. 6. Classi ica ion o biosenso s acco ding o he ype o ansduce . A.-B. Bl´ azquez and N. Jim´ enez de Oya Syn he ic and Sys ems Bio echnology 10 (2025) 194–206 198 and a o dabili y [57]. Ano he good ea u e is ha hese de ices ha e he possibili y o being coupled wi h o he biosensing echniques o enhanced de ec ion. All hese cha ac e is ics make elec ochemical biosenso s a good pla o m o be used in a wide spec um o applica ions anging om moni o ing wa e [57], biomedical diagnos ics [58], ood analysis [59], o pa hogen de ec ion [60,61]. 1.4.3.2.2. Op ical biosenso s. Op ical biosenso de ec ion elies on he in e ac ion be ween op ical echnologies wi h a bio ecogni ion elemen . They ha e ecei ed conside able a en ion in ecen decades as powe ul de ec ion and analysis ools wi h b oad applica ions, as hey p esen impo an ad an ages compa ed o o he well-es ablished biosenso echnologies, such as noise educ ion and immuni y o elec- omagne ic in e e ence [62]. Op ical biosensing can be classi ied in o wo main ca ego ies: label- ee and label-based. As p e iously men ioned in elec ochemical biosenso s, in label- ee de ec ion he signal o igina es di ec ly om he in e ac ion be ween he analyzed sample and he ansduce , while label-based de ec ion employs a ag [63]. Based on he de ec ion p inciple, hese de ices can be classi ied as hose ha measu e luminescence, luo escence, colo changes, abso - bance, e lec ance, o luo escence emissions ha occu in he ul a i- ole (UV), isible, o nea -in a ed (NIR) spec al anges [64]. Table 3 summa izes he mos commonly used op ical echniques in e ms o hei de ec ion mechanism. These biosenso s ha e demons a ed aluable e icacy in he de ec- ion o biological analy es and ha e shown no able ad ances in hei use in biomedicine [65,66], ood sa e y [67,68], pa hogen de ec ion [69], and he bio echnology indus y [70,71]. 1.4.3.2.3. The mome ic biosenso s. The mome ic biosenso s, also known as calo ime ic, quan i y hea changes in a sample and i s en i- onmen . These biosenso s a e c ea ed by immobilizing he bio ecep o in a empe a u e senso , which de ec s and measu es he ene ge ic al- e a ions, such as hea exchange, p oduced in he analy e [72]. The echnique is a ailable o he analysis o any eac ion ha gene a es a measu able amoun o hea . In his sense, he wide use ulness o calo- ime ic biosenso s is based on he ac ha all biochemical eac ions a e associa ed wi h a change in hea , ei he gene a ing o abso bing hea . Consequen ly, a single calo ime ic ansduce can se e as a e sa ile pla o m o quan i y mul iple bioma ke s [73]. The e o e calo ime ic biosenso s a e used in a wide ange o applica ions, such as ood p o- cessing and sa e y [73], pa hogen de ec ion [74], clinical moni o ing [75,76], o en i onmen al de e mina ions [77]. 1.4.3.2.4. Mass-based biosenso s. Mass-based biosenso s, also called g a ime ic, eac o a small a ia ion in he mass o he binding analy e gene a ing a de ec able signal [78]. The mos commonly used g a i- me ic ansduce s a e hin piezoelec ic qua z c ys als ha esona e a a pa icula equency in esponse o bo h he applied cu en and he mass o he de ec ed ma e ial [38]. These piezoelec ic biosenso s s and ou as op imal ools, as hey acili a e apid, label- ee, eal- ime de ec ion o analy es wi hou equi ing speci ic eagen s o complex sample manipula ions. Acous ic biosenso s a e a ype o piezoelec ic de ices ha use he acous ic wa es gene a ed by hese ma e ials o iden i y he a ge analy e h ough induced changes in he ea u es o he acous ic wa e [79]. Mass-based biosenso s a e impo an in he de elopmen o minia- u ized, po able de ices o pes icide de ec ion [80], i us de ec ion [81], ood p ocessing echnologies [82], o medical diagnosis [83], among o he s. 1.4.3.2.5. Magne ic biosenso s. A magne ic biosenso is a de ice able o ans o m a magne ic ield in o an elec ical signal. In ecen yea s, hese biosenso s ha e been inc easingly used in he de elopmen o biosenso s hanks o he special cha ac e is ics o magne ic ma e ials. The gene al p ocedu e o biological de ec ion using a magne ic biosenso in ol es ini ially immobilizing he p obe on he senso su ace and subsequen ly allowing he sample, which con ains magne ic labels, o low ac oss he su ace o he senso [84]. Magne ic nanopa icles (MNPs) ha e ecen ly eme ged as sui able labels o he de elopmen o his echnology, enabling he de ec ion and iden i ica ion o a huge a ie y o physical, chemical, and biological agen s [85]. Magne ic biosenso s a e widely applied o moni o biological in- e ac ions and apid de ec ion o analy es as POCT, mainly in d ug disco e y [86], i us de ec ion [87], biomedical applica ions [88,89], o ood analysis [90]. 1.5. Biosenso s o la i i us de ec ion As men ioned abo e, he diagnosis o la i i us in ec ions is usually pe o med by adi ional me hods, mainly se ology and molecula as- says. Howe e , hese echniques ha e a se ies o disad an ages, such as he high economic bu den ha makes diagnos ic es s una o dable in low-income coun ies, whe e he impac o la i i uses is usually impo an . O he d awbacks a e he need o quali ied pe sonnel and he ac ha hey a e ime-consuming me hods. In ecen yea s, hese ob- s acles a e being o e come hanks o he de elopmen o biosenso s as new, as , and sensi i e me hods in he diagnosis o la i i uses (Fig. 7). Biosenso s o diagnosing la i i us o e no able ad an ages o e con en ional me hods, pa icula ly hei abili y o p oduce easy handling po able de ices. Mos echniques allow o di ec analysis o samples wi hou equi ing any p e- ea men since he mos commonly used samples include se um, sali a, and o he body luids om pa ien s. These samples acili a e easy handling and apid esul s. Some imes, biosenso samples equi e p io p ocessing, ypically ollowing he same p ocedu es used in con en ional diagnos ic me hods, such as nucleic acid ex ac ion o simila echniques [91]. Howe e , i is essen ial o conside ha biosenso s also ha e some d awbacks such as po en ial s abili y issues wi h he componen s o al e a ion in pa hogen de ec ion due o mu an i uses (Table 4). 1.5.1. Biosenso s o he de ec ion o dengue i us DENV is a se ious global public heal h conce n a ec ing mo e han 90 million cases and app oxima ely 40,000 dea hs pe yea . Cu en ly, many esea che s ha e explo ed biosenso s as a no el al e na i e ech- nology o de ec he i us o he p esence o an ibodies. This app oach o e s se e al ad an ages, including sensi i i y, cos -e ec i eness, easy p oduc ion, apid esul s wi h quan i a i e analysis, and he possibili y o de eloping POCT de ices [100]. A la ge majo i y o esea che s ha e de eloped elec ochemical biosenso s o he diagnosis o DENV, mainly based on he elec o- chemical impedance spec oscopy (EIS) echnique [101]. In his sense, a Table 3 Op ical biosenso s. De ec ion mechanism Fluo escence Phospho escence Re lec ion UV/Vis/IR abso bance F¨ o s e Resonan Ene gy T ans e (FRET) In e e ome y Su ace Plasmon Resonance (SPR) A.-B. Bl´ azquez and N. Jim´ enez de Oya Syn he ic and Sys ems Bio echnology 10 (2025) 194–206 199 huge numbe o di e en elec odes ha e been designed, he mos common being hose made o g aphene and gold. G aphene elec odes ha e been used ecen ly due o hei cha ac e is ics o imp o ed sensi- i i y, hus achie ing low de ec ion limi s. A g aphi e-based DNA biosenso was de eloped speci ically o iden i y he DENV-3 se o ype [102]. Addi ionally, ano he me hod was in oduced o de ec he dengue i us, capable o disc imina ing be ween he di e en se o ypes, using an elec ochemical me hod based on g aphene polyme [103]. Gold elec odes a e also widely used in DENV biosenso s. Luna e al. [104] immobilized he lec in concana alin A on he gold elec ode. This app oach was also employed by Oli ei a e al. wi h se a om in ec ed pa ien s who de eloped dengue e e (DF) o dengue hemo hagic e e (DHF) [105]. In his case, a ia ions in cha ge ans e esis ance we e u ilized o di e en ia e he senso esponses o he se a examined ( om pa ien s wi h DF o DHF), he eby aiding in he disc imina ion o he s ages o se e i y o he disease. Resea che s ha e also used o he di e en lec ins immobilized on gold elec odes, such as C amoll, iden i ied om C a ylia mollis seeds [106,107], o Bauhinia monand a lec in (BmoLL) [96] o he de ec ion o DENV-1, DENV-2, and DENV-3 se o ypes. Mos elec ochemical biosenso s o he de ec ion o DENV ha e been de eloped a ge ing he non-s uc u al p o eins (NS) o he i us as a bio ecep ion elemen . Di e en s udies indica e ha NS1 an igen is abundan in he se um o pa ien s du ing he ea ly s ages o DENV in ec ion [108,109], making i a po en ial ma ke o acu e dengue i us in ec ion. Immunosenso s a ge ing his p o ein ha e been p oduced wi h di e en elec odes. In his sense, Pa kash e al. de eloped an elec ochemical immunosenso modi ied wi h he s ep a idin/bio in sys em on sc een-p in ed ca bon elec odes (SPCEs) o he de ec ion o he NS1 an igen. The biosenso was es ed in pa ien se um samples [110]. NS1 de ec ion sys em was also de eloped by Junio e al. [111], using a DNA ap ame , and o he immunosenso s based on sc een-p in ed elec odes we e de eloped by di e en au ho s [112–114]. Cecche o e al. also de eloped di e en capaci i e elec- ochemical me hods o he de ec ion o NS1 in human samples [92, 115]. Simila app oaches ha e been used wi h an i-DENV2 IgG o o he an ibodies immobilized on nanopo ous alumina elec odes [116,117]. The use o DENV DNA p obes has also been widely exploi ed as bio ecep ion elemen s in he de elopmen o elec ochemical bio- senso s. In his ega d, Shingai e al. c ea ed a biosenso whe e he DNA was immobilized on he su ace o a ZnO/P –Pd nanocomposi es elec- ode [118]. Di e en DNA p obes we e also assessed by many o he au ho s [119–121]. Mo e ecen ly, CRISPR-based de ec ion app oaches ha e been Fig. 7. Ad an ages and disad an ages o classical me hods e sus biosenso s applied in he de ec ion o la i i uses. Table 4 Ad an ages, disad an ages and de ec ed a ge s p esen ed acco ding o he p incipal la i i us diagnos ic app oaches. Me hods Ad an ages Disad an ages De ec ed a ge s Re . Classical me hods RT-PCR Sensi i i y Expensi eness Time-consuming Fla i i us RNA molecula de ec ion [29] Only applicable in ea ly s ages o in ec ion qRT-PCR Sensi i i y Quan i a i e esul s Expensi eness Quali ied pe sonel equi ed Fla i i us RNA molecula de ec ion [29] Only o ea ly s ages o in ec ion ELISA De ec ion in ea ly and la e s ages o in ec ion C oss- eac i i y Fla i i us an ibody de ec ion (IgM/ IgG) [30] Low speci ici y PRNT Gold-s anda d me hod o la i i uses BSL-3 acili ies Fla i i us neu alizing an ibodies de ec ion [23] Quali ied pe sonel equi ed Only o ea ly s ages o in ec ion Cell cul u e Vi al isola ion capabili y BSL-3 acili ies Fla i i us isola ion [23] Quali ied pe sonel equi ed Only o ea ly s ages o in ec ion Biosenso s (based on bio ecep o s) Enzyme A o dable S abili y challenges DENV [56] Easy handling Speci ici y An ibody High speci ici y P ope immobiliza ion o an ibodies DENV [92] Sensi i i y ZIKV [93] Po able JEV [94] WNV [95] Nucleic acid A o dable Limi a ions in de ec ion o mu a ed i uses DENV [96] Speci ici y Nucleic acid ex ac ion equi ed ZIKV [97, 98]Low sensi i i y JEV   WNV [99] A.-B. Bl´ azquez and N. Jim´ enez de Oya Syn he ic and Sys ems Bio echnology 10 (2025) 194–206 200 de eloped as a sensi i e me hod o e eal he p esence o DENV in di e en samples, such as blood and sali a [122], o RNA samples [123, 124]. Al hough elec ochemical biosenso s a e he mos commonly used in he de ec ion o DENV, he e is also a wide a ie y o app oaches ha use op ical biosenso s. As happened wi h elec ochemical biosenso s, di e en p ocedu es ha e been de eloped. In his sense, i al RNA has been e alua ed by Chen e al. [125] wi h gold nanopa icles coupled o qua z c ys als. O he au ho s ha e pe o med di e en RNA biosenso s [126–129]. Among op ical biosenso s, he use o an ibodies in he de elopmen o immunosenso s is a echnique also exploi ed o he de ec ion o DENV. Di e en immunosenso s based on su ace plasmon esonance (SPR) ha e been designed o DENV IgM an ibody de ec ion [130–132] o he iden i ica ion o dengue NS1 an igens [133]. A ias e al. de el- oped a diagnos ic ool based on a chemiluminescen op ical ibe immunosenso (OFIS), o he de ec ion o an i-DENV immunoglobulin M (IgM) in human se um samples [134]. Mass-based biosenso s ha e also been desc ibed o he de ec ion o DENV. In his case, he mos commonly used a e piezoelec ic de ices such as immunosenso s ha de ec i al E o NS1 p o eins [135–137], o nucleic acid biosensing [125]. Di e en app oaches ha e been used by au ho s o de e mine and compa e he sensi i i y and ecogni ion capabili ies o biosenso s, hus con i ming he de ec ion o his la i i us. These me hods include echniques such as ELISA, he use o p e iously i a ed i uses o comme cially a ailable p o ein s anda ds, among o he s. Despi e g ea e o s o de elop DENV biosenso s, only a ew ha e been comme cialized. Comme cially a ailable de ices a e Vi oT ack Dengue Acu e, capable o de ec ing dengue NS1 an igen, an impo an bioma ke o ea ly DENV in ec ion [138], and Bioline™ DENGUE DUO, which de ec bo h DENV NS1 and an i-DENV speci ic IgM/IgG an i- bodies [139]. As p e iously men ioned, he main cha ac e is ics o DENV bio- senso s should be po abili y, low cos , and easy handling o make hem ideal de ec ion sys ems o POCT and ield applica ions. Likewise, he abili y o dis inguish be ween di e en se o ypes and he po en ial o ea ly de ec ion o in ec ion make biosenso s o DENV a highly e ec i e ool in pa hogen diagnosis. 1.5.2. Biosenso s o he de ec ion o Zika i us ZIKV is a ela i ely ecen i us iden i ied in he mid-20 h cen u y. Hence, s udies ca ied ou on he de elopmen o biosenso s o i s de ec ion a e sca ce. The i us can cause se ious diseases such as e al mic ocephaly o Guillain-Ba ´ e synd ome. Since mos in ec ions occu in de eloping coun ies, he e is an u gen need o a o dable and e ec i e biosenso s capable o apidly and accu a ely iden i ying ZIKV in epidemic a eas [19]. In he sea ch o elec ochemical biosenso s, di e en pla o ms ha e been used, such as an immunosenso based on ZnO nanos uc u es immobilized wi h ZIKV-NS1 an ibody [140] o he immobiliza ion o p o ein E wi h he de elopmen o quan um do s in combina ion wi h sc een-p in ed ca bon elec odes [141]. Using elec- ochemical impedance spec oscopy and squa e wa e ol amme y, a biosenso capable o disc imina ing ZIKV an ibodies in blood and sali a om DENV i us-speci ic an ibodies was also assessed [142]. A g aphene-enabled biosenso was c ea ed o de ec ZIKV wi h a speci ic NS1 monoclonal an ibody [97]. Likewise, he elec ochemical modi i- ca ion o pencil ca bon g aphi e elec odes [143], o he de ec ion o genomic RNA using a new pla o m based on g aphi e elec odes ha e been used [91]. O he ele an elec ochemical echniques a e he de elopmen o impedance elec ical sensing assay on pape mic ochips [144] o he immobiliza ion o su ace imp in ed polyme s o sensi i e and speci ic de ec ion o ZIKV [145]. Label- ee biosenso s ha e also been desc ibed, such as an impedi- me ic elec ochemical DNA genosenso [146] o an E p o ein-based immunosenso [93]. Among op ical biosenso s, some au ho s ha e used colo ime y o he de elopmen o di e en pla o ms showing high speci ici y in he de ec ion o ZIKV [93]. Ano he wo k desc ibed he de elopmen o localized su ace plasmon esonance echnology o de ec he NS1 p o- ein in an immuno luo escence biosenso [147]. Mo eo e , a mass-based biosenso has been desc ibed o he de ec ion o ZIKV using suscep ome y measu emen echniques [148]. As men ioned in he case o DENV biosenso s, a ious s a egies ha e been employed by esea che s o assess and compa e he sensi i i y and ecogni ion capabili ies o de ices, he eby con i ming he de ec ion o his la i i us [91,140]. Howe e , u he esea ch is needed o achie e apid and accu a e iden i ica ion using biosensing echnologies in he case o ZIKV [149]. 1.5.3. Biosenso s o he de ec ion o Wes Nile i us The esea ch cu en ly being ca ied ou in he de elopmen o biosenso s o he de ec ion o WNV is e y limi ed. As happened wi h o he la i i uses, assays a e mainly aimed a he de elopmen o elec- ochemical biosenso s. In his sense, Pa k e al. applied an al e na ing cu en elec o he mal low echnology o p o ide a apid biosenso pla o m based on WNV DNA ap ame s exhibi ing high selec i i y [150]. O he genosenso s using DNA ha e also been desc ibed [99]. On he o he hand, di e en assays based on su ace-enhanced Raman sca e ing (SERS) echnology ha e been epo ed o WNV de ec ion. An immunoassay o he de ec ion o DNA o he pa hogen was desc ibed using Au nanopa icles [151]. These echniques enable apid and sensi i e de ec ion o WNV, hus con ibu ing o he diagnosis and con ol o he i us. Label- ee biosenso s o WNV de ec ion ha e also been desc ibed based on capaci i e echniques [152] o using a pape -based mic o- luidic analy ical de ice wi h in eg a ed mic owi e Au elec odes [153]. These biosenso s can de ec complemen a y DNA agmen s o i al pa icles in a apid and low-cos way, making hem sui able o POCT de ices. In he case o op ical biosensing, esea ch was ca ied ou o de elop a ibe op ic immunosenso o he de ec ion o an i-WNV IgG an ibodies in se um [95]. 1.5.4. Biosenso s o he de ec ion o Japanese encephali is i us JEV ou b eaks p edominan ly a ec u al egions. The e o e, i is no easible o es ablish complex labo a o y acili ies and deploy ained echnicians o i s diagnosis. E o s in ad ances in diagnos ic echniques aim o c ea e as e , cos -e ec i e, and mo e sensi i e me hods o de ec JEV [154]. These inno a ions, including nano echnology, a e being in- eg a ed in o biosenso s o enhance hei sensi i i y, he eby acili a ing highly e ec i e de ec ion mechanisms. Elec ochemical biosenso s ha e been epo ed o he de ec ion o JEV. A de ice consis ing o ca bon nanopa icles modi ied SPCEs was assessed using cyclic ol amme y (CV) and EIS o de ec JEV an igens in se um samples [155]. Rela ed p ocedu es we e based on gold [98] o sil e [94] nanopa icles modi ied SPCE. O he elec ochemical s a e- gies ha e been employed such as gold-coa ed magne ic beads [156], g aphene de i a i es [157,158], o su ace-enhanced Raman spec oscopy-based biosenso s [159]. Label- ee-based echniques ha e also been desc ibed. In his ega d, wo elec ochemical immunosenso s based on an i-JEV IgG an ibodies immobilized on di e en polyaniline mic oelec odes ha e been e- po ed o he de ec ion o JEV an igens [160,161]. Ano he epo ed label- ee biosenso was based on he immobiliza ion o JEV-speci ic se um an ibodies on a silanized su ace o an in e digi a ed senso [162]. Rega ding op ical biosenso s, Liang e al. p oduced a luo escen senso based on i us-molecula imp in ed polyme s ancho ed on he su ace o silica [163], while He e al. designed a luo escen senso based on i us-imp in ed polyme s [164]. The luo escence in ensi y was enhanced in he i s wo k by he luo escence esonance ene gy A.-B. Bl´ azquez and N. Jim´ enez de Oya Syn he ic and Sys ems Bio echnology 10 (2025) 194–206 201 ans e (FRET) echnique. Likewise, o he luo escence molecula ly imp in ed senso s based on di e en amewo ks we e desc ibed [165, 166], showing ema kable selec i i y and sensi i i y in de ec ing JEV. 1.5.5. Biosenso s o he de ec ion o yellow e e and ick-bo ne encephali is i uses Ongoing esea ch in o he de elopmen o biosenso s o he de ec- ion o YFV and TBEV is a he limi ed. Only a ew wo ks desc ibe he de elopmen o biosenso s used in he diagnosis o YFV in human se um o plasma samples [167,168], and no de ices ha e been de eloped o he speci ic de ec ion o TBEV. Howe e , biosenso s ha e been de el- oped o he diagnosis o mul iple la i i uses, including hese wo pa hogens. 1.5.6. Biosenso s o mul iple la i i us de ec ion As p e iously men ioned, c oss- eac i i y be ween la i i uses is equen , pa icula ly in egions wi h i al co-ci cula ion. The wide- sp ead dis ibu ion o mosqui oes ac ing as ec o s p omo es he coex- is ence o la i i al in ec ions in o e lapping egions. Mos la i i uses exhibi signi ican s uc u al simila i ies, igge ing a c oss- eac i e immune esponse ha can esul in alse posi i es in con en ional se ological es s, especially in seconda y in ec ions. To aid in i us ecogni ion, biosenso s capable o dis inguishing be ween hem ha e been de ised. The mos common de ices a e hose designed o he concu en de ec ion o ZIKV and DENV, using di e en app oaches o hei de elopmen , such as he applica ion o DNA-nano echnology-based de ec ion biosenso s, he de elopmen o elec ochemical de ices wi h di e en wo king elec odes o each i us, o he use o CRISPR ech- nology, among o he s [169–171]. The iden i ica ion o DENV and YF has been conduc ed wi h a mul- iplexed pa hogen de ec ion pla o m using mul i-colo ed sil e nano- pla es [172]. Ano he complex biosenso has been designed o di e en ia e DENV, ZIKV, and YFV in ec ions. A omic o ce mic oscopy analyses alida ed he elec ode su ace modi ica ion and un eiled a ied opog aphy h oughou he bio ecogni ion p ocess. CV and EIS we e used o he cha ac e iza ion o he biosenso [173]. Fo he diagnosis o TBEV, a bi-pa ame ic se ological mic oa ay was de eloped o de ec TBEV and WNV. The de ec ion sys em was based on he speci ic sequen ial de ec ion o an ibodies [174]. De ec ion o TBEV, ZIKV, YFV, and JEV, as well as o he ela ed a bo i uses, has been desc ibed using a mul iplex ecombinase polyme ase ampli ica ion-based nucleic acid de ec ion pla o m. The op imal con- di ions enable luo escence de ec ion o nucleic acids wi h high eloci y, speci ici y, and sensi i i y. Fu he mo e, a low-cos , easy- o-handle POCT de ice was enginee ed o isualiza ion [175]. A comme cially a ailable es is he SD Biosenso STANDARD Q A bo Panel I (Z/D/C/Y). The es consis s o a ch oma og aphic immunoassay o he de ec ion o ZIKV, DENV, and YFV in human se um, plasma, o whole blood (h ps://www.sdbiosenso .com/p oduc /p oduc _ iew? p oduc _no=219). 2. Conclusions Fla i i uses (genus O ho la i i us) a e a bo i uses (a h opod-bo ne i uses) ansmi ed mainly by mosqui oes o icks. This genus includes mul iple well-known human, animal, and zoono ic pa hogens. The spec um o symp oms induced by la i i us in ec ions anges om asymp oma ic o mild e e o se e e mani es a ions, mos ly hemo - hagic o neu ological complica ions, which can ul ima ely lead o dea h. Due o a ious ac o s, such as he globaliza ion o a el and ade, clima e change, al e a ions in land use, and changes in ec o beha io , se e al la i i uses a e eme ging as signi ican global heal h conce ns, expanding hei p esence o new habi a s no p e iously colonized [176]. The e a e cu en ly no speci ic an i i al ea men s o la i i uses, and only a limi ed numbe o accines ha e been app o ed o human use agains some o hem. Hence, unde s anding he biology o la i i uses and de eloping apid and sensi i e diagnos ic es s is essen ial o p e en he sp ead o hese po en ially li e- h ea ening pa hogens. Fla i i us in ec ions a e usually diagnosed by con en ional me hods, p edominan ly se ology and molecula assays. Howe e , hese ech- niques ha e se e al d awbacks, including high cos s, making diagnos ic es s una o dable in low-income coun ies whe e he impac o la i- i us is signi ican . Mo eo e , hese me hods equi e quali ied pe sonnel and a e ime-consuming. I is wo h men ioning ha c oss- eac i i y be ween la i i uses is equen , especially in a eas whe e mul iple i- uses ci cula e simul aneously. The wide dis ibu ion o mosqui oes, which ac as ec o s, acili a es he co-occu ence o la i i al in ec ions in o e lapping geog aphical a eas. Many la i i uses sha e no able an igenic simila i ies, leading o a c oss- eac i e immune esponse ha can p oduce alse posi i es in se ological es s. To add ess his challenge, he de elopmen o biosenso s has been o e coming hese obs acles in ecen yea s, showing new, apid, and sensi i e app oaches o he diagnosis o la i i uses. In his sense, biosenso s o e a wide ange o ad an ages such as excep ional sensi i i y and speci ici y easy handling, low cos , and he abili y o p o ide apid esponses and pe o m eal- ime analyses [101]. All hese ea u es acili a e apid in e en ion in he e en o heal h eme gencies such as pandemic si ua ions. As p e iously men ioned, mos echniques enable he di ec analysis o samples wi hou he need o p e- ea men , as he commonly u ilized samples—such as se um, sali a, and o he bodily luids—allow o s aigh o wa d handling and quick esul s. Howe e , in some cases, biosenso samples may equi e p io p ocessing, usually employing me hods simila o hose used in con en ional diagnos ics, such as nucleic acid ex ac ion and o he ela ed echniques. Fu he mo e, he possibili y o designing compac -sized po able de ices ende s biosenso s ideal o poin -o -ca e es ing (POCT) in bioanaly ical clinics [154]. Cu en ly, he e is a g owing demand o POCT o swi ly de ec in ec ious diseases, including hose caused by i uses. These de ices a e c ucial o slowing he sp ead o in ec ious diseases by enabling eal- ime es ing and p o iding apid, high-quali y diagnoses, as la i i us ou b eaks occu mainly in u al a eas, making i un easible o ha e specialized labo a o ies and skilled wo ke s o ca y ou diagnoses. Hence, biosenso s a e he mos no able ad ance in he de ec ion o hese li e- h ea ening pa hogens. In his sense, many e- sea che s ha e de eloped se e al ypes o equipmen classi ied acco d- ing o he echnology used by hei design. In he case o la i i uses, mos o he de ices de eloped use elec ochemical ansduce echnol- ogy, combined wi h a huge a ie y o bio ecep o s, hus achie ing a signi ican numbe o de ices wi h di e en speci ici y and sensi i i y o he apid and e icien diagnosis o he a o emen ioned i us. Since DENV is he mos signi ican li e- h ea ening la i i us, causing app oxima ely 40,000 dea hs each yea , biosensing echnolo- gies a e p ima ily ocused on he ea ly de ec ion o his pa hogen. Some s udies ha e shown ha he dengue i us nons uc u al 1 (NS1) an igen is p esen in he se um o pa ien s du ing he ea ly s ages o in ec ion, indica ing ha NS1 may se e as an e ec i e ma ke o acu e dengue i us in ec ion. In his con ex , biosenso s designed o de ec DENV NS1 could p o ide a eliable means o iden i ying ea ly acu e dengue in- ec ions, he eby po en ially imp o ing disease, as no speci ic ea - men s a e a ailable o dengue o any o he la i i us. The exis ing ea men op ions a e only suppo i e and ocused on mi iga ing com- plica ions and educing he se e i y o symp oms. On he o he hand, biosenso s in la i i us esea ch a e enabling e ec i e disc imina ion be ween ela ed s ains, o e en, in he case o DENV, be ween se o ypes. O pa icula impo ance is o highligh ha ein ec ions wi h a ious se o ypes o his i us can exace ba e he disease, po en ially leading o a al ou comes due o an ibody-dependen enhancemen (ADE) [177]. In his con ex , ecen ad ancemen s in biosenso echnology o la i i us a e ocused on achie ing accu a e in ec ion diagnosis. No ably, new de ices a e being de eloped ha A.-B. Bl´ azquez and N. Jim´ enez de Oya Syn he ic and Sys ems Bio echnology 10 (2025) 194–206 202