Development of Simple and Rapid Bead-Based Cytometric Immunoassays Using Superparamagnetic Hybrid Core-Shell Microparticles

De elopmen o Simple and Rapid Bead-Based Cy ome ic Immunoassays Using Supe pa amagne ic Hyb id Co e−Shell Mic opa icles Cha lie Tobias, Daniel López-Pue ollano, An onio Abad-Somo illa, Josep V. Me cade , An onio Abad-Fuen es, and Knu Ru ack* Ci e This: ACS Meas. Sci. Au 2024, 4, 678−688 Read Online ACCESS Me ics & Mo e A icle Recommenda ions ABSTRACT: Flow cy ome y-based immunoassays a e aluable in biomedical esea ch and clinical applica ions due o hei high h oughpu and mul ianaly e capabili y, bu hei adop ion in a eas such as ood sa e y and en i onmen al moni o ing is limi ed by long assay imes and complex wo k lows. Rapid, simpli ied bead-based cy ome ic immunoassays a e needed o make hese me hods iable o poin -o -need applica ions, especially wi h he inc easing accessibili y o minia u ized cy ome e s. This wo k in oduces supe pa amagne ic hyb id polys y ene-silica co e−shell mic opa icles as p omising al e na i es o con en- ional polyme beads in compe i i e cy ome ic immunoassays. These beads, ea u ing high speci ici y, sensi i i y, and excellen handling capabili ies ia magne ic sepa a ion, we e e alua ed wi h h ee di e en an ibodies and binding me hods, showing a ia ions in signal in ensi y based on he an ibody and i s a achmen me hod. The op imal pe o mance was achie ed h ough a seconda y an ibody binding app oach, p o iding s ong and consis en signals wi h minimal unce ain y. The op imized p o ocol made i possible o achie e a de ec ion limi o 0.025 nM in a o al assay ime o only 15 min and was success ully used o de ec och a oxin A (OTA) in aw lou samples. This wo k highligh s he po en ial o hese beads as e sa ile ools o low cy ome y-based immunoassays, wi h signi ican implica ions o ood sa e y, animal heal h, en i onmen al moni o ing, and clinical diagnos ics. KEYWORDS: an ibody-based, bead-based assay, co e−shell pa icles, cy ome y, myco oxins ■INTRODUCTION Flow cy ome y-based compe i i e immunoassays a e widely used in biomedical esea ch and clinical applica ions o quan i y and de ec di e en analy es in complex biological samples, since hey combine he high- h oughpu po en ial o immunoassays wi h he mul ianaly e capabili y o liquid ch oma og aphy (LC) echniques. 1−3 Al hough such ap- p oaches also hold p omise in ields like ood sa e y, animal heal h, and en i onmen al moni o ing, hey a e only slowly inding hei way in o hese a eas. 4−12 On he one hand, his is due o he he e ogenei y o he low molecula weigh a ge analy es and he limi ed a ailabili y o an ibodies o hem, bu on he o he hand also o he usually a he long assay imes and mul is ep wo k lows, which o en ake 1 h o longe due o (se e al) incuba ion s eps and hus canno e en compe e wi h LC me hods in e ms o ime o esul . Howe e , simplici y and speed would gene ally be desi able i immunoanaly ical me hods we e o make he s ep o poin -o -need applica ions, o which hey a e gene ally well sui ed due o he ad an age ha obus low cy ome e s become inc easingly a ailable; o ins ance, o esou ce-limi ed se ings, 13−15 hei measu emen pe iphe y can be minia u ized 7,16 and as e mix-and- ead immunoassay o ma s a e being de eloped. 9 Cen al as a model analy e o he p esen wo k is och a oxin A (OTA), a myco oxin commonly ound in g ain p oduc s, co ee, cacao, g apes, and po k, posing se e e heal h isks. 17−19 To add ess hese isks, he Eu opean Union (EU) has ins i u ed maximum le els (ML) o OTA ac oss a ious oods u s. 20 While se e al me hods exis o OTA analysis, immunochemical and ch oma og aphic echniques domina e. Among he i s g oup, enzyme-linked immunoso ben assays (ELISA) and la e al low immunoassays (LFIA) a e widely used, 21,22 wi h se e al ki s being al eady a ailable on he ma ke . Rega ding ch oma og aphic echniques, high-pe o m- ance liquid ch oma og aphy (HPLC) combined wi h luo- escence de ec ion, a e immunoa ini y column cleanup, is he Recei ed: July 9, 2024 Re ised: Augus 9, 2024 Accep ed: Sep embe 4, 2024 Published: Sep embe 17, 2024 A iclepubs.acs.o g/measu eau © 2024 The Au ho s. Published by Ame ican Chemical Socie y 678 h ps://doi.o g/10.1021/acsmeasu esciau.4c00038 ACS Meas. Sci. Au 2024, 4, 678−688 This a icle is licensed unde CC-BY 4.0 Downloaded ia CSIC on Decembe 23, 2024 a 12:41:00 (UTC). See h ps://pubs.acs.o g/sha ingguidelines o op ions on how o legi ima ely sha e published a icles. ecommended app oach by he Eu opean Commi ee o S anda diza ion. 23 Ne e heless, o en mass spec ome y de ec ion (MS/MS) is p e e ed o OTA de e mina ion a ace le els. 24,25 While ELISA and LFIA a e known o hei obus ness, hey ha e d awbacks wi h ega d o speed and handling s eps (ELISA) as well as he capabili y o mul iplexing o o p ocess a high numbe o samples (LFIA), making hem sui able o ou ine analysis o a limi ed se o myco oxins and/o samples. Ch oma og aphy-based echni- ques a e p e e ed o egula o y asks ha equi e highe accu acy, sensi i i y, o a mul ianaly e app oach, ye such analyses a e slow and cos ly. As a po en ial al e na i e, as men ioned abo e, bead-based immunoassays u ilizing low cy ome y o e a lo o ad an ages. Howe e , only ew examples ha e been desc ibed o OTA, using ela i ely hea y pa icles in all cases and equi ing long assay imes. 26−29 As we a e in e es ed in de eloping cy ome ic and mic o luidic immunoassays o on-si e use, we s a ed some ime ago o de elop a modula pa icle pla o m ha should e en ually lead o a sui able app oach. The pla o m uses polys y ene-silica co e−shell beads 30,31 which ha e also been success ully used by o he s 32 and a e cu en ly being comme cialized. 33 In addi ion, we ha e ecen ly ex ended his pla o m by inco po a ing magne ic ea u es. 34 These hyb id beads consis o a polys y ene co e and a silica shell in which magne ic nanopa icles a e embedded, acili a ing handling du ing washing and e en ion in analy ical assays. The ou e silica su ace can be easily modi ied by silane chemis y so ha an ibodies o o he molecules o in e es can be a ached. In addi ion, he pa icle co e can be doped wi h luo escen dyes, enabling use in mul iplexed assays o simul aneous de ec ion o mul iple a ge s. 35 Compa ed o pa icles doped wi h me allic nanopa icles o gi e hem magne ic p ope ies, hese hyb id beads a e ligh e , esul ing in slowe sedimen a ion and making hem mo e sui able o low cy ome y. 36−38 These p ope ies enable e icien analysis o la ge sample olumes, as hea y pa icles can lead o sedimen a ion, clogging, signal sa u a ion, and op ical in e e - ences, which a ec s da a quali y and ins umen pe o mance in low cy ome y. When ligh e pa icles a e a ailable as an op ion, hey a e gene ally p e e ed o a oid hese p oblems and ensu e mo e accu a e low cy ome y measu emen s. 39 In he p esen s udy, we used a compe i i e immunoassay app oach wi h a luo escen compe i o o de ec OTA in o de o e alua e he applicabili y o hese new ma e ials in cy ome y assays. By quan i ying he amoun o compe i o bound o he beads, we we e able o de e mine he OTA concen a ion, which allowed o p ecise and accu a e de ec ion wi hou a washing o isola ion s ep. Ou second objec i e was o e alua e he unc ionaliza ion p o ocol o he supe pa amagne ic hyb id co e−shell beads. Fo his pu pose, we in es iga ed h ee di e en an i-OTA an ibodies ha had also been p e iously p oduced by ou g oup. 40 In addi ion, h ee me hods o immobilizing he p ima y an ibodies on he bead su ace we e es ed, namely, di ec binding o an ibodies o he bead su ace, binding ia p o ein G, and binding ia a seconda y goa -an imouse (GAM) an ibody. Finally, by employing he op imal assay con igu a ion, we analyzed eal lou samples ob ained om a mill o OTA, ealizing a low limi o de ec ion in a a o able o e all assay ime. ■MATERIALS AND METHODS Poly( inylpy olidone) (PVP10, 10 kDa, Sigma), s y ene (Sigma), basic alumina (Al2O3, B ockmann I, Ac os), and azo-biscyano ale ic acid (ACVA, MP Biomedicals) we e used o he PVP-coa ed polys y ene co e syn hesis. FeCl3·6H2O (AppliChem) and FeCl2· 4H2O (Bake ) we e used o he p epa a ion o magne ic nano- pa icles. Te ae hoxyo hosilica e (TEOS, Me ck) and ammonia solu ion (NH3, 32%, Supelco) we e used o he silica coa ing. (3- Aminop opyl) ie hoxysilane (APTES, Ald ich) was used o amino unc ionaliza ion and succinic anhyd ide (Me ck) o la e ca boxylic acid unc ionaliza ion. 1-E hyl-3-[3-(dime hylamino)p opyl]- ca bodiimide hyd ochlo ide (EDC, Me ck) and N-hyd oxysul osucci- nimide sodium sal (sNHS, Sigma) we e used o pa icle ac i a ion. A iniPu e Goa An i-Mouse (GAM) IgG, Fcγ agmen speci ic, unconjuga ed seconda y an ibody om Jackson ImmunoResea ch, and Pie ce Recombinan P o ein G we e used o u he pa icle unc ionaliza ion. Phospha e-bu e ed saline (PBS, pH 7.4, 10 mM, 130 mM NaCl), 2-(N-mo pholino)e hanesul onic acid bu e (MES, pH 6.0, 10 mM), and bica bona e bu e (pH 9.6, 50 mM) we e p epa ed in Milli-Q g ade wa e . T is bu e was p epa ed using 10 mM T is−HCl (pH 7.5), 120 mM NaCl, 20 mM CaCl2, and 40 mM MgCl2in Milli-Q g ade wa e . The OTA luo escein compe i o conjuga e (OTA-F) and he OTA s anda d we e pu chased om Aokin. Monoclonal an i-OTA an ibodies we e p e iously p oduced and cha ac e ized by ou g oup. 40 Measu emen s we e conduc ed ia low cy ome y using a BD Accu i C6 ins umen equipped wi h 488 and 640 nm lase s o exci a ion and included he eco ding o he o wa d sca e (FSC) and sidewa d sca e (SSC) signals o he pa icles a angles o 180 and 90°, espec i ely. Addi ionally, he luo escence signal in he FL1 channel (488 nm, 533/30.H il e ) was cap u ed. To de e mine he IC50 alues, he da a ob ained om he compe i i e assay we e analyzed and plo ed in he O igin so wa e (O iginLab) using a ou - pa ame e sigmoidal i ing. Fo SEM imaging, he pa icles we e dispe sed in e hanol and subjec ed o ul asonica ion o 5 min. To p epa e he samples o analysis, hey we e d op-cas ed on o con en ional ca bon TEM g ids. Imaging o indi idual pa icles was pe o med using a Zeiss Sup a 40 scanning elec on mic oscope (Zeiss), equipped wi h a high- esolu ion ca hode (Scho ky ield emi e ), an E e ha −Tho nley seconda y elec on (SE) de ec o , and an SE InLens de ec o . Fo ansmission elec on mic oscopy mode (TSEM o STEM-in-SEM), a dedica ed “ ansmission” sample holde was u ilized. Syn hesis o Hyb id Pa icles The supe pa amagne ic mic opa icles we e p epa ed by a ou e adap ed om e 34, elabo a ing ou ea lie a chi ec u e desc ibed in e s 30,31,35 o he p esen one wi h polys y ene (PS) as he co e, i on oxide (Fe3O4) nanopa icles as a magne izable in e laye , and an ou e silica shell o p o ec ion and u he unc ionaliza ion. The syn hesis o he PS pa icles was ca ied ou ia dispe sion polyme iza ion by eac ing a solu ion o 170 mg o PVP10 in 10 mL o E OH wi h 1 mL o s y ene, il e ed h ough basic aluminum oxide, in a glass ial a e lushing he mix u e wi h a gon o 30 min and subsequen ly ini ia ing i by he addi ion o 0.5 mL o a solu ion o 105 mg o ACVA in 10 mL o MeOH, lushed wi h a gon, unde s i ing a 70 °C in an a gon a mosphe e o e nigh . The esul ing pa icles we e cen i uged, washed wi h wa e and E OH mul iple imes, and hen d ied a oom empe a u e. Syn hesis o Supe pa amagne ic I on Oxide Nanopa icles (SPIONs) To syn hesize SPIONs, 0.465 g o FeCl3·6H2O and 0.172 g o FeCl2· 4H2O we e dissol ed in 100 mL o Milli-Q wa e in a ound-bo om lask. The solu ion was pu ged wi h a gon o 20 min be o e slowly adding a solu ion o 4 g o PVP10 in 58 mL o NH3solu ion (16%). The eac ion mix u e was s i ed a 150 pm o 1.5 h using a mechanical s i e . The pa icles we e hen washed se e al imes wi h wa e using magne ic sepa a ion and s o ed in a e ige a o a a concen a ion o app oxima ely 3% (w/ ) in Milli-Q wa e . ACS Measu emen Science Au pubs.acs.o g/measu eau A icle h ps://doi.o g/10.1021/acsmeasu esciau.4c00038 ACS Meas. Sci. Au 2024, 4, 678−688 679 Coa ing o PS Co es wi h SPIONs (SPION@PS) The PS co es we e coa ed wi h a laye o SPIONs by suspending 60 mg o PS co es and 2 mL o Fe3O4pa icles (3% in wa e ) in 30 mL o Milli-Q wa e in Falcon ubes. The coa ing p ocess was ca ied ou by placing he ubes on a o a o pla e a 40 pm o 1.5 h. A e wa d, he pa icles we e washed wice wi h wa e and once wi h e hanol, using magne ic sepa a ion, be o e d ying. Coa ing o SPION@PS Pa icles wi h Silica Shell (SiO2@SPION@PS) 555 μL o NH3solu ion (32%) was added o a dispe sion o 60 mg o SPION@PS pa icles in 30 mL o E OH and 1 mL o Milli-Q wa e while s i ing a 150 pm using a mechanical s i e . Then, 555 μL o TEOS was added d opwise. The mix u e was s i ed o e nigh a 38 °C, ollowed by mul iple washes wi h wa e and E OH, using magne ic sepa a ion. Finally, he pa icles we e d ied a oom empe a u e. Func ionaliza ion o SiO2@SPION@PS wi h Amino G oups Acco ding o a p e iously epo ed p o ocol, 35 he SiO2@SPION@PS pa icles we e unc ionalized wi h amino g oups by i s ac i a ing he pa icles’ su ace a e suspending 20 mg o he pa icles in 800 μL o E OH, adding 400 μL o 1 M HCl in E OH, and sonica ing he mix u e in a sonica ion ba h o 5 min. A e wa d, he pa icles we e washed wice wi h 400 μL o E OH and edispe sed in 400 μL o E OH. Fo amino modi ica ion, 8 μL o APTES was added o he pa icle dispe sion and he mix u e was allowed o eac in a he momixe (800 pm) a 40 °C o e nigh . Subsequen ly, he pa icles we e washed h ee imes wi h a mix u e o E OH:H2O in a 1:1 a io, be o e d ying in acuum a oom empe a u e. Re unc ionaliza ion o SiO2@SPION@PS wi h Ca boxylic Acid G oups To modi y he su ace o he ma e ials wi h ca boxylic acid g oups o acile biomolecule a achmen , 5 mg o he co esponding amino- modi ied pa icles was dispe sed in 1.5 mL o absolu e E OH in a 2 mL Eppendo ube. A solu ion o 30 μL 10% w/ o succinic anhyd ide in dime hyl o mamide was added o he pa icle dispe sion, and he mix u e was hen allowed o eac in a he momixe (800 pm) a 40 °C o e nigh . A e wa d, he pa icles we e washed h ee imes wi h a mix u e o E OH:H2O in a 1:1 a io. In he inal s ep, 500 μL o e hanol was added o he pa icles o ob ain a s ock solu ion wi h a inal concen a ion o 1% (w/ ). Coupling o P o ein G and GAM o Re unc ionalized SiO2@SPION@PS F om he 1% (w/ ) s ock solu ions, 10 μL o SiO2@SPION@PS pa icles was dispe sed in 100 μL o MES bu e . To his pa icle dispe sion, 80 μL o a eshly p epa ed solu ion o EDC and 160 μL o s-NHS solu ion, bo h in MES bu e and wi h a concen a ion o 1% (w/ ), we e added. The mix u e was incuba ed a oom empe a u e o 15 min, and hen 50 μL o ei he p o ein G (2.4 mg mL−1) o GAM (2.4 mg mL−1) was added and incuba ed o e nigh on a o a o pla e a 40 pm and oom empe a u e. A e wa d, he pa icles we e washed wice wi h 500 μL o PBS and edispe sed in 200 μL o esh PBS o each a s ock solu ion concen a ion o 0.05% (w/ ). Di ec Binding o he P ima y An ibody Fo he di ec binding o he an i-OTA mAbs, he p e iously desc ibed p o ocol o p o ein G o GAM was ollowed. In each case, 20 μL a 1 mg mL−1in PBS o each o he mAbs was used. The washing was pe o med as p e iously desc ibed o he cap u e p o ein-modi ied pa icles. Coupling o he P ima y An ibodies o he P o ein G- and GAM-Modi ied Pa icles A suspension con aining 0.035% p o ein G o GAM-modi ied pa icles was p epa ed in 300 μL o PBS, and hen 20 μL o solu ions con aining he mAbs a 1 mg mL−1in PBS was added. The mix u e was incuba ed on a o a o pla e o 1 h a oom empe a u e. Subsequen ly, he pa icles we e washed once wi h PBS and he olume was es o ed o he o iginal 200 μL, yielding a inal pa icle concen a ion o 0.05% (w/ ). Inhibi ion Tes s The inhibi ion es s we e pe o med in la -bo omed PS 96-well pla es. To do so, 10 μL (0.05%) o unc ionalized pa icles was placed in o indi idual wells con aining 100 μL o PBS. Nex , 5 μL o OTA-F a di e en concen a ions (0.1, 0.01, 0.001, and 0.0001 μM plus a blank con ol) was added. The same p ocedu e was epea ed o a second se o wells, bu wi h he addi ion o 5 μL o a 1 μM OTA solu ion. The pla e was incuba ed unde gen le shaking o 15 min. The inhibi ion a e (in pe cen ) was calcula ed as he quo ien o he signals (mean coun s in FL1) measu ed o he wells con aining OTA (subsc ip xOTA) and he wells wi hou OTA (subsc ip noOTA), ep esen ing he maximum signal, using he same OTA-F concen- a ion in all wells: Inhibi ion a e FL FL1 / 1 xOTA noOTA = Compe i i e Assays In he compe i i e assay, 10 μL (0.05%) o pa icles was dispensed in o indi idual wells o a well pla e along wi h 100 μL o PBS bu e . To ini ia e he assay, 5 μL o OTA (a di e en concen a ions, including a blank) was added o he wells. Subsequen ly, 5 μL o OTA-F was in oduced in o each well, and he assay was gen ly shaken o 10 min in he da k. Finally, he well pla e was subjec ed o measu emen using a low cy ome e wi h a measu emen ime o 1 min o a single well. Sample Ex ac ion 2 g o sample was placed in a 15 mL ube along wi h 8 mL o CH2Cl2 and 200 μL o H3PO46M. The ube was hen placed on a o a o pla e o 15 min a 40 pm and hen cen i uged a 8000 pm o 10 min. F om he esul ing mix u e, 1 mL o he CH2Cl2phase was ans e ed o a 2 mL Eppendo ube and 500 μL o bica bona e bu e was added. The Eppendo ube was mixed on a o a o pla e o 10 min, la e cen i uged a 6000 pm o 3 min, and he aqueous phase was collec ed. Finally, he ex ac was dilu ed in assay bu e (PNa75, phospha e bu e pH 7.4, 75 mM) using a dilu ion ac o o 1/25. Unce ain y Budge Calcula ions The ba ch- o-ba ch ep oducibili y o he syn hesized pa icles is gi en by he coe icien o a ia ion CV, as de e mined ia he o wa d sca e ing pa ame e measu ed wi h he low cy ome e , esul ing in CV sc= 5% o he PS co e, CV sc= 7% o he pa icles a e he i on oxide coa ing s ep and CV sc= 13% a e silica coa ing. The coupling o an ibodies o SiO2@SPION@PS accoun s o an addi ional combined unce ain y o u el T1=3%, including a) Weighing o 5 mg o he co esponding amino- unc ionalized pa icles (balance Me le Toledo lab 205 ±0.01 mg) u el a= 0.2% b) Dispe sing he pa icles in 500 μL o E OH a e eac ion wi h succinic anhyd ide (Eppendo e e ence pipe 500 μL±3μL) u el b= 0.6% c) Dilu ing 10 μL o he suspension in 100 μL o MES bu e (Eppendo e e ence pipe es ±0.1 μL o 10 μL and ±0.8 μL o 100 μL pipe ) u el c= 1.8% d) Adding 80 μL o a solu ion o EDC (1%) and 160 μL o a solu ion o NHS (1%) u el d=1.3% e) Adding 50 μL o ei he GAM o p o ein G o he solu ion; u el e = 1% ) Cen i uging and washing (2×) and esuspending in 200 μL PBS; u el = 0.6% g) Adding 20 μL o he di e en mAbs u el = 1.5% The unce ain ies o he p epa a ion and execu ion o he assay include dilu ions o he s ock solu ions o he analy e, i.e., dilu ing 5 μL o s ock solu ion in 100 μL o bu e (u el V= 2%), u he dilu ing s ock solu ions o OTA in wa e o ob aining s anda d OTA o OTA- F, in PBS successi e dilu ion o he mo he solu ion: n×u el V,nmax = 9, mixing o 10 μL o pa icle suspension (0.05%) and 100 μL o bu e ACS Measu emen Science Au pubs.acs.o g/measu eau A icle h ps://doi.o g/10.1021/acsmeasu esciau.4c00038 ACS Meas. Sci. Au 2024, 4, 678−688 680 + 5 μL o OTA and 5 μL o OTA-F (u el a= 4.8%) and he con ibu ions om he cy ome e measu emen s: a) Rela i e unce ain y o coun s o pa icles egis e ed: u el cy ≤ 1.5% b) Expe imen al s anda d de ia ion o eplica e measu emen s: u el ≤2% amoun ing o a o al ela i e unce ain y o u el T= 5.8% o he assay. ■RESULTS AND DISCUSSION Ou s udy add esses he applica ion o a nex gene a ion o magne ic hyb id mic opa icles o he de e mina ion o small- molecule analy es in apid cy ome y assays using monoclonal an ibodies as he de ec ion en i y. While he p esen wo k demons a es he pe o mance o he app oach wi h he use case o de ec ing OTA in whea lou ex ac s, he pa icle a chi ec u e al eady includes a u u e use o he beads in mul iplexed au oma ized assays. In addi ion, he aim was o u he exploi a s aigh o wa d mix-and- ead app oach as ou beads a e ideally sui ed o use wi h many di e en an ibodies wi hou he need o change he p o ocol o he beads. Exis ing magne ic pa icles ace challenges ela ed o s abili y, unc ionaliza ion, and weigh (sedimen a ion). To o e come hese limi a ions, we ecen ly imp o ed ou p omising hyb id pa icle pla o m, being ad an ageous in e ms o s abili y, p ac ical weigh , acile unc ionaliza ion, and cus omizable su ace a ea, 30,31,35 wi h ano he ea u e, a magne ic unc ion- ali y. 34 The p esen wo k epo s on he unc ionaliza ion, deploymen , and pe o mance o hese beads in analy ical assays, o e ing ad an ages in e ms o e iciency and eliabili y compa ed o p e ious app oaches. The design a ionale o he supe pa amagne ic co e−shell mic opa icles and hei s epwise syn hesis is as ollows. Poly( inylpy olidone)-s abilized (PVP10, a e age molecula weigh 10 kDa) PS mic opa icles cons i u e he co e (Figu e 1a, (i)), because hey can be acilely p epa ed in sizes well sui able o single-pa icle assays (1−3μm) wi h high monodispe si y and he possibili y o dope hem subsequen ly wi h o ganic dyes ia a simple swelling p ocedu e. 31 A hin laye o SPIONs ha does no co e he su ace comple ely is hen coa ed on o he PS co es o endow magne ic p ope ies while a oiding sedimen a ion issues and lea ing space o any op ical moie ies doped in o he co e o be exci ed in an applica ion (Figu e 1a, (ii)). Finally, a closed seconda y silica shell is coa ed on o he i s shell o p o ec ion and u he acile unc ionaliza ion ia silane chemis y (Figu e 1a, (iii)). The esul ing pa icles as obse ed in a scanning elec on mic oscope (SEM) a e shown in Figu e 2. They ha e an o e all diame e o 1.8 ±0.1 μm. The silica shell, measu ing ca. 30 nm in hickness, inco po a es 5 nm SPIONs. The image in Figu e 2a shows he whole pa icle, while he image in Figu e 2b highligh s he su ace a ea and i s oughness. To isualize he SPION laye , TSEM images we e aken as seen in he image in Figu e 2c. Fi s , hese pa icles ha e a simila su ace a ea o comme cial mic opa icles o analy ical applica ions (e.g., Dynabeads, The mo Fishe ) while p esen ing no able ad an ages: Poly inylpy olidone (PVP) se es as a s abilize Figu e 1. (a) Wo k low o bead p epa a ion: coa ing o PS beads (i) wi h SPIONs (ii) and an insula ing SiO2shell (iii); he amino unc ionaliza ion and ca boxy e unc ionaliza ion s eps a e omi ed o simplici y. Binding o an ibodies o beads h ough one-s ep di ec a achmen (i , ou e DA) o in wo s eps ia p o ein G ( , ou e PG) o goa an imouse Ab ( , ou e GAM). (b) Wo k low o mix-and- ead assay: pipe ing o h ee solu ions in o wells ( i), shaking ( ii), and cy ome ic measu emen and analysis ( iii). ACS Measu emen Science Au pubs.acs.o g/measu eau A icle h ps://doi.o g/10.1021/acsmeasu esciau.4c00038 ACS Meas. Sci. Au 2024, 4, 678−688 681 in he pa icles, no only o e ing a long- e m s abili y o he SPIONs by hemsel es 41 bu also acili a ing a s aigh o wa d deco a ion wi h SPIONs be o e o e g ow h o an insula ing silica coa ing. They a e ligh e in weigh han con en ional silica o PS pa icles con aining he magne ic i on oxide nanopa icles in he mic obead’s co e, 10,12 making hem mo e sui able o longe use in a ious applica ions. The pa icles’ dis inc i ely ex u ed, high-su ace a ea s uc u e can be a ibu ed o he p esence o SPIONs pa ially deco a ing he pu e PS co e. This laye , combined wi h a dense silica coa ing, esul s in a oughe su ace, as p e iously desc ibed. 34 An impo an new aspec o hese pa icles is his ype o laye o med by he SPIONs. Al hough hey a e no magne ic ou side a magne ic ield due o he size o he nanopa icles, he en i e co e−shell pa icle can easily be mo ed by a magne . The p esence o a a he loose laye ins ead o a closed shell o nanopa icles also enables op ical encoding o he co es, since a dense shell o nanopa icles would esul in op ically opaque co e pa icles. 35 Las ly, he silica coa ing no only p o ides mul iple a enues o unc ionaliza ion bu also ac s as a p o ec i e ba ie , enabling he pa icles o be used in acidic en i onmen s whe e i on would ypically oxidize and lose i s magne ic p ope ies. In ou case, his silica ou e shell was employed o include amino g oups h ough classical silane chemis y using APTES. These amino g oups we e la e con e ed in o ca boxylic acid g oups, ia succinic anhyd ide, o he u he immobiliza ion o he p o eins. To de elop an easy- o-handle assay p o ocol using he supe pa amagne ic hyb id PS-co e magne ic silica-shell beads, se e al es s we e conduc ed. These es s included he binding o di e en p o eins, such as p ima y monoclonal an ibody, seconda y polyclonal an ibody, o p o ein G o he pa icle su ace (Figu e 1a, (i ) and/o ( )), as well as he in es iga ion o an ibody in e ac ions wi h he selec ed luo escen compe i o and he a ge analy e, OTA. Conside ing ou p e ious expe ience wo king wi h his myco oxin, h ee di e en monoclonal an i-OTA an ibodies we e selec ed o e alua ion (e#115, #223, and b#311; labeling ollows he labeling in e 40). These h ee an ibodies we e selec ed because hey we e able o in e ac wi h a compe i o o OTA p epa ed h ough he same posi ion as he luo escen luo escein-con aining compe i o (OTA-F) selec ed o his s udy. Figu e 3 shows he s uc u e o OTA, OTA-F, and he hap ens ha we e p e iously used o an ibody gene a ion. Each o he hap ens was used o he gene a ion o one o he an ibodies es ed he e, as indica ed by he hap ens and he le e in he an ibody code. To bind he di e en p o eins o he pa icle su ace, we used EDC/sNHS chemis y o o m co alen amide bonds be ween he amino g oups o he lysine esidues o he p o eins wi h he ca boxylic acid g oups on he su ace o he pa icles. Addi ionally, he pu pose o using p o ein G (Figu e 1a, ou e PG) o GAM (Figu e 1a, ou e GAM) as assis ing cap u e p o eins was o e alua e he impo ance o he o ien a ion o he p ima y an i-OTA an ibody. This was done wi h he aim o minimize s a is ical e o s, ensu ing ha he p ima y an ibody would consis en ly ha e he co ec o ien a ion (pa a ope acing he analy e) on he pa icles. As a con ol, he p ima y an i-OTA an ibodies we e also di ec ly a ached (Figu e 1a, ou e DA) o he su ace using he same EDC/sNHS app oach. Conside ing he inal pa icle assay applica ion, low cy ome y was chosen as he p e e ed me hod o all es ing due o i s use - iendly ea u es, including he a ailabili y o an au osample and apid assay eadou (Figu e 1b). The luo escence emi ed by he OTA-F compe i o on he su ace o he pa icle is cen e ed a 518 nm and was de ec ed h ough a 533/30.H bandpass il e be o e co ela ion wi h he FSC signal o dis inguish i om he excess o compe i o s ill in solu ion. In a p elimina y s udy, o es he capabili y o hese an ibodies o ecognize he OTA-F compe i o , h ee immobiliza ion app oaches ia ou es DA, PG, and GAM we e e alua ed o he h ee an i-OTA an ibodies. The maximum signal om he su ace o a single pa icle was measu ed in he absence o OTA (mos OTA-F bound), while also a p onounced inhibi ion a e was assessed o a ela i ely Figu e 2. SEM images ((a) ull iew and (b) close-up o su ace) and TSEM images ((c) close-up o su ace) o he polys y ene-co e magne ic silica-shell beads. ACS Measu emen Science Au pubs.acs.o g/measu eau A icle h ps://doi.o g/10.1021/acsmeasu esciau.4c00038 ACS Meas. Sci. Au 2024, 4, 678−688 682 high concen a ion o OTA (40 nM), ca ying ou hese expe imen s o all combina ions. The inhibi ion a e is de ined he e as he quo ien o he signals in he wells wi h OTA and he signals in he wells wi hou OTA, wi h all wells ha ing he same OTA-F concen a ion; see Ma e ials and Me hods o de ails. A his poin , he concen a ion o an ibody was kep cons an a 300 ng o an ibody pe μg o pa icle. Figu e 4 shows he g aphs ep esen ing he inhibi ion a es as do ed lines and he maximum signals as ba s o he nine ypes o pa icles used in he s udy. The le y-axis deno es he maximum signal achie able wi h he compe i o o each ype o binding, while he igh y-axis ep esen s he inhibi ion o he compe i o in he p esence o 40 nM OTA. To ensu e he lowes a e o alse-nega i e esul s, i was c ucial o employ he lowes possible concen a ion o compe i o . Excess luo es- cence om he le o e compe i o could po en ially lead o inaccu a e ou comes ia unspeci ic binding. The highes inhibi ions we e obse ed when an ibody e#115 was di ec ly bound o he pa icles ( ou e DA, 80%) o when an ibody #223 was used in conjunc ion wi h GAM ( ou e GAM, 85%). Con e sely, hese es ing s eps e ealed ha an ibody b#311 showed no signi ican in e ac ion wi h he compe i o , p obably due o he lack o a spa ial linke be ween he OTA and luo escein subuni s, as epo e s wi h longe linke s we e able o in e ac wi h his an ibody in ELISA. 21 The e o e, an ibody b#311 was excluded om u he in es iga ions he e. To de e mine he op imal signal- o-noise a io, we e alua ed di e en concen a ions o compe i o wi h he o he wo an ibodies. The bes a io was achie ed wi h a concen a ion o 0.01 μM o OTA-F. While an ibody e#115 displayed he highes o e all signal, i s inhibi ion a e eached 80% in he case o an ibody binding along ou e DA. On he o he hand, an ibody #223 exhibi ed success ul pe o mance wi h he seconda y an ibody a achmen ia ou e GAM and an imp o ed inhibi ion a e o 84%, aligning wi h he objec i e Figu e 3. S uc u es o (a) OTA, (b) luo escen compe i o OTA-F, and (c) hap ens used o an ibody gene a ion. Figu e 4. Maximum signal o he bound compe i o in he absence o analy e in he assay (le y-axis) and inhibi ion a e o a concen a ion o 40 nM o OTA ( igh y-axis) o (a) an ibody e#115, (b) an ibody #233, and (c) an ibody b#311 a ached ia ou es DA, PG, and GAM o he i le beads. ACS Measu emen Science Au pubs.acs.o g/measu eau A icle h ps://doi.o g/10.1021/acsmeasu esciau.4c00038 ACS Meas. Sci. Au 2024, 4, 678−688 683 o his p ojec . Consequen ly, u he es ing in ol ed a compa ison o e#115/DA and #223/GAM. To gain mo e insigh in o he an ibody immobiliza ion s ep, we decided o de e mine he equi ed amoun o an ibody o maximum co e age o he pa icles. In ha ega d, he assay wi h an ibody e#115 was chosen o his e alua ion since i was pe o ming be e in he DA app oach. To accoun o any unce ain ies ega ding he beha io o he an ibody on he su ace, ideally ull unc ionaliza ion o he su ace is desi able. Because o ha , we conduc ed u he es s using wo di e en concen a ions o his an ibody: 300 ng o an ibody μg−1o pa icle, a simila concen a ion o he one employed in he p elimina y es s, and 450 ng o an ibody μg−1 o pa icle. The calib a ion cu es we e gene a ed by es ing di e en concen a ions o OTA in he p esence o a 0.01 μM compe i o concen a ion. Figu e 5 (bo om) shows ha bo h concen a ions ul ima ely yielded simila signal in en- si ies, indica ing ha he maximum binding capaci y o he pa icles’ su ace had been eached wi h he lowe concen- a ion es ed. The hal inhibi o y concen a ion (IC50) commonly iden i ied as he concen a ions a he in lec ion poin when he minimal asymp o e ends o ze o o hese wo assays we e de e mined o be 1.4 and 1.7 nM o he concen a ions o 300 and 450 ng o an ibody μg−1o pa icle, espec i ely. Conside ing he simila beha io o bo h concen a ions shown, he lowes one was selec ed o u he op imiza ion. Since an ibody #223 was showing a be e inhibi ion in he p elimina y es when immobilized ia ou e GAM, his assay was chosen o u he de elopmen . In he same way as be o e, wo di e en an ibody concen a ions we e es ed, 300 and 450 ng o an ibody μg−1o pa icle. A his poin , i mus be no ed ha he pa icles we e p e iously unc ionalized wi h a la ge excess o cap u e an ibody GAM (960 ng o seconda y an ibody μg−1o pa icle) o sa u a e he pa icle su ace and elimina e he a ailabili y o ac i e places as a limi ing ac o . Figu e 5 ( op) p esen s a signal inc ease wi h he amoun o p ima y an ibody #223 used, sugges ing ha he amoun o cap u e an ibody is adequa e o he es ed concen a ions o monoclonal an i- body. The e o e, he decisi e ac o in selec ing he op imal assay was a comp omise be ween an adequa e maximal signal and minimal IC50. The IC50 alues o he wo an ibody concen a ions es ed, 300 and 450 ng o an ibody μg−1o pa icle, we e 0.7 and 1.0 nM, espec i ely. The ac ha he maximum speci ic binding (Bmax) and IC50 a e di e en ly a ec ed by a change in an ibody concen a ion o ou e GAM compa ed o ou e DA, i.e., a educ ion in Bmax wi h simila IC50 o ou e GAM compa ed o simila Bmax and IC50 o ou e DA, is p esumably due o he di e en ways o an ibody a achmen . As men ioned abo e, an ibodies e#115 a e co alen ly ancho ed o he su ace o he beads in ou e DA and bo h concen a ions esul ed in a i ually iden ical unc ionaliza ion densi y, leading o a e age FL1 signals o 3310 ±60 coun s and a 20% highe IC50 o he highe an ibody concen a ion. Howe e , wi h ou e GAM, wo biomac omolecules a e sequen ially bound o he su ace o he beads, so ha no only he co alen unc ionaliza ion densi y wi h GAM is decisi e, bu also he nonco alen binding o #223 o GAM plays a ole. Ob iously, he occupa ion o he GAM uni s by #223 is no ye quan i a i e a he lowe concen a ion, which leads o a 25% highe Bmax a he 450 ng o an ibody μg−1o pa icle. In addi ion, IC50 is also 45% highe o he highe an ibody concen a ion. As lowe IC50 alues co espond o imp o ed de ec ion limi s and he absolu e signal in ensi ies measu ed a e su icien o all assay combina ions, he op imized assay u ilized he lowe an ibody amoun o 300 ng o an ibody μg−1o pa icle. We made addi ional e o s o enhance he assays by expe imen ing wi h pos an ibody a achmen washing s eps and modi ying he bu e composi ion. Howe e , hese a emp s yielded no disce nible imp o emen . The in oduc ion o an addi ional washing s ep wi h bu e , in addi ion o cen i uga ion and sol en exchange, did no lead o any no iceable changes in assay signals. Simila ly, ansi ioning om a PBS o a TRIS-based bu e did no esul in an inc ease o dec ease in signal in ensi y beyond measu emen unce ain y. Thus, ad an ageously, he mix-and- ead app oach as shown in Figu e 1b p o ed o yield op imum pe o mance while being simple and as . To assess he ep oducibili y o he de eloped assay, i s day- o-day pe o mance was s udied. The assay was pe o med on ou sepa a e days, wi h ou di e en ba ches o beads, wi h h ee eplica es each, yielding he calib a ion cu es p esen ed in Figu e 6. Figu e 6 p o ides a di ec compa ison o he wo pa icle- based immunoassays, i.e., in he op panel, OTA was again de ec ed wi h an ibody e#115 a ached o he su ace o he pa icles along ou e DA, while in he bo om panel, he combina ion #223/GAM was used. The igu e shows ha he de ec ion limi s exhibi ed minimal a iabili y h oughou he 4- Figu e 5. Calib a ion cu es using e#115/DA ( op) and #223/GAM (bo om) in di e en amoun s o de e mine he maximum co e age o he pa icles. ACS Measu emen Science Au pubs.acs.o g/measu eau A icle h ps://doi.o g/10.1021/acsmeasu esciau.4c00038 ACS Meas. Sci. Au 2024, 4, 678−688 684 day es ing pe iod, and wi h an IC50 o 0.1 nM, he assay in ol ing he use o GAM and monoclonal an ibody #223 appea s o be he mos e ec i e. The la ge e o obse ed in pa icles using an ibody e#115 is likely due o he di ec binding me hod used in hei p epa a ion. Wi hou con ol o e he o ien a ion o he pa a ope egion, he unce ain y in measu ing lowe concen- a ions o OTA is mainly in luenced by andom e o s due o he lack in di ec ionali y a he han he p epa a ion o he ca ie pa icles o he assay p o ocol. The limi o de ec ion (LoD, acco ding o DIN 32645:2008- 11) achie ed by ou assay using he seconda y an ibody is 0.025 nM, which is in he lowe ange o cy ome y assays o OTA epo ed in he li e a u e. 26−29 An essen ial imp o emen in compa ison o comme cial assays is he o e all assay ime. The en i e p ocess akes less han 15 min, 10 min o incuba ion plus ans e ime o he low cy ome e , and ca. 1 min measu emen ime o a cy ome y un, in con as o 1 h o mo e o mos epo ed examples, 4−7,10,12 he main imp o emen being he simplici y and g ea ly educed incuba ion ime. The abili y o de ec low concen a ions o he a ge analy e u he demons a es he obus ness and eliabili y o ou de eloped assay. No ably, he achie ed de ec ion limi is compa able o ha o many immunochemical me hods, such as ELISA, and e en ou pe o ms ce ain echniques like elec o- chemical analysis. 21,42,43 The ela i ely low and consis en e o obse ed in ou measu emen s ensu es compliance wi h he myco oxin es ing limi s se by he Eu opean Union (EU). 20 The e sa ili y o he analy ical me hod desc ibed in his s udy allows o i s applicabili y in a ious pa icle-based assays, only limi ed by he a ailabili y o a compe i o and i s pe o mance wi h he a ailable an ibodies. Fo u he p oo , we conduc ed a eal-li e example by es ing whea lou ob ained om a local mill. The lou sample was ob ained di ec ly om he mill wi hou any u he p ocessing, e lec ing he ypical handling a he acili y. To ensu e eliable measu emen s, a po ion o he sample was spiked wi h OTA. The ini ial concen a ion o OTA was known h ough a egula analysis by Eu o ins, commissioned by he mill. We spiked he samples wi h an OTA s anda d, esul ing in a concen a ion o 6.1 μg kg−1, while he pu e lou sample con ained 0.2 μg kg−1, a alue below he maximum limi es ablished by he EU. 20 Following an ex ac ion me hod p e iously desc ibed based on he acidic cha ac e is ics o OTA, 21 we pe o med he assay and ob ained alues o 0.4 ± 0.1 μg kg−1 o he blank lou sample and 6.4 ±0.1 μg kg−1 o he spiked sample. This esul ed in a eco e y a e o 104%, which alls wi hin he commonly accep ed ange o 90−110%. We a emp ed al e na i e ex ac ion me hods, including he use o e hanol as he ma ix, bu un o una ely, hese e o s did no yield any meaning ul esul s. Using e hanol only in he ex ac ion s ep, no signal was de ec ed, and when measu e- men s we e conduc ed in e hanol, a ma ix e ec was obse ed, ul ima ely esul ing in inconclusi e ou comes. The success ul eco e y demons a es he accu acy and e iciency o his assay and u he highligh s i s sui abili y o p ac ical applica ions. Addi ionally, he speed o he assay shows by equi ing only a 10 min incuba ion ollowed by measu emen s comple ed in less han 30 s. The assay p esen ed in ou s udy demons a es a compe i i e pe o mance o OTA de ec ion compa ed o exis ing assays, in e ms o bo h de ec ion limi and analysis ime. The o al ime equi ed o ou assay is less han 11 min pe sample, which is on pa wi h he pe o mance o LFIAs. 21,22,44,45 Unlike LFIAs, which ypically canno pe o m pa allel analyses, ou assay u ilizes a well pla e au osample wi h a cy ome e , allowing o apid sequen ial analysis. This se up p o ides a signi ican ad an age in e ms o h oughpu and speed and has he ad an age o ha ing much be e mul iplexing po en ial han LFIAs due o he PS co e, which can be easily encoded wi h dyes. While ELISAs gene ally o e he capabili y o p ocess mul iple samples simul aneously, making hem sui able o high- h oughpu scena ios, hey a e commonly cha ac e ized by signi ican ly longe analysis imes while showing com- pa able de ec ion limi s. 21,46,47 Also, he use o o he binde s such as nanobodies 48,49 o ap ame s 50 o he employmen o no el ampli ica ion s a egies 51−53 has no changed he si ua ion wi h espec o assay ime and has also no led o d ama ic imp o emen s in de ec ion limi s. Compa ed wi h ELISAs, ou assay o e s a much as e al e na i e wi hou sac i icing sensi i i y. The assay de eloped he e is especially ad an ageous in si ua ions equi ing immedia e decision- making, like he p ocessing o a shipmen in a mill. I would also be bene icial in labo a o ies whe e he h oughpu o LFIA is needed wi h highe sensi i i y and speci ici y. Figu e 6. Calib a ion cu es o he inal op imized assays; assay based on combina ion an ibody e#115/DA ( op) and assay based on combina ion an ibody #223/GAM (bo om). ACS Measu emen Science Au pubs.acs.o g/measu eau A icle h ps://doi.o g/10.1021/acsmeasu esciau.4c00038 ACS Meas. Sci. Au 2024, 4, 678−688 685 Wi h he ease and speed o his assay, u he wo k in ou labo a o ies add esses mul iplexed analysis, allowing o he de ec ion o mul iple oxins simul aneously. ■CONCLUSIONS Ou p esen wo k ocused on he de elopmen and op imiza ion o a pa icle-based immunoassay o he de ec ion o och a oxin A. By employing no el pa icles wi h imp o ed s abili y, easy unc ionaliza ion, and low p ac ical weigh , we we e able o o e come he limi a ions o exis ing magne ic pa icles and de elop a as mix-and- ead immunoassay o ma wi h an o e all assay ime o <15 min. Flow cy ome y p o ed o be a sui able me hod o he measu emen o he assay, o e ing use - iendly handling and quick eadou . The calib a ion cu es ob ained h ough he assay demons a ed he maximum binding capaci y o he pa icles and he impac o di e en an ibody a achmen and concen a ions. Day- o-day measu emen s showed consis en calib a ion cu es wi h minimal a ia ion especially o he ou e employing a seconda y goa -an imouse an ibody o he deco a ion o he pa icles wi h he p ima y an ibody. No ably, he limi o de ec ion o he inal op imized assay, using he seconda y an ibody, was de e mined o be 0.025 nM, su passing he de ec ion limi s o many exis ing me hods, including elec ochemical ones. Fu he mo e, he de eloped assay demons a ed i s p ac icali y and applicabili y in a eal-li e scena io by success ully analyzing a lou sample ob ained om a mill and de ec ing OTA wi h an excellen eco e y a e. This unde sco es he ease and e iciency o he p oposed assay, making i a aluable ool o apid myco oxin es ing in a ious ood indus ies. Wi h i s e sa ili y, po en ial o mul iplexing, and sui abili y o simpli ied luidic sys ems due o he magne ic p ope ies o he pa icles employed, his assay holds p omise o he de ec ion o o he oxins o low molecula weigh pollu an s as well. The esul s ob ained in his s udy con ibu e o he ad ancemen o myco oxin de ec ion me hods and o e p ac ical solu ions o ood sa e y analysis. ■AUTHOR INFORMATION Co esponding Au ho Knu Ru ack −Chemical and Op ical Sensing Di ision, Bundesans al u Ma e ial o schung und -p u ung (BAM), Be lin D-12489, Ge many; o cid.o g/0000-0002-5589- 5548; Email: [email p o ec ed] Au ho s Cha lie Tobias −Chemical and Op ical Sensing Di ision, Bundesans al u Ma e ial o schung und -p u ung (BAM), Be lin D-12489, Ge many; o cid.o g/0000-0003-2440- 3181 Daniel López-Pue ollano −Chemical and Op ical Sensing Di ision, Bundesans al u Ma e ial o schung und -p u ung (BAM), Be lin D-12489, Ge many; Depa men o O ganic Chemis y, Uni e si y o Valencia, Bu jasso , Valencia 46100, Spain; o cid.o g/0000-0001-7259-4918 An onio Abad-Somo illa −Depa men o O ganic Chemis y, Uni e si y o Valencia, Bu jasso , Valencia 46100, Spain; o cid.o g/0000-0002-5599-3682 Josep V. Me cade −Ins i u e o Ag icul u al Chemis y and Food Technology (IATA), Spanish Council o Scien i ic Resea ch (CSIC), Pa e na, Valencia 46980, Spain; o cid.o g/0000-0002-1838-2647 An onio Abad-Fuen es −Ins i u e o Ag icul u al Chemis y and Food Technology (IATA), Spanish Council o Scien i ic Resea ch (CSIC), Pa e na, Valencia 46980, Spain; o cid.o g/0000-0001-5672-1438 Comple e con ac in o ma ion is a ailable a : h ps://pubs.acs.o g/10.1021/acsmeasu esciau.4c00038 Au ho Con ibu ions CRediT: Cha lie Tobias concep ualiza ion, o mal analysis, in es iga ion, me hodology, isualiza ion, w i ing - o iginal d a ; Daniel Lopez-Pue ollano o mal analysis, unding acquisi ion, in es iga ion, me hodology, p ojec adminis a ion, isualiza ion, w i ing - e iew & edi ing; An onio Abad- Somo illa me hodology, esou ces, w i ing - e iew & edi ing; Josep Vicen Me cade me hodology, esou ces, w i ing - e iew & edi ing; An onio Abad-Fuen es me hodology, esou ces, w i ing - e iew & edi ing; Knu Ru ack concep ualiza ion, unding acquisi ion, me hodology, p ojec adminis a ion, supe ision, isualiza ion, w i ing - e iew & edi ing. No es The au ho s decla e no compe ing inancial in e es . ■ACKNOWLEDGMENTS This wo k has been pe o med as a pa o BAM’s Focus A ea P ojec “MamaLoCA�Modula , mul iplexed, an ibody-based lab-on-chip analyse o ood con ol” o which inancial suppo by BAM is acknowledged (No. TF20). D.L.-P. was suppo ed by a Ma ga i a Salas pos doc o al g an (MS21-068) om he Minis e io de Uni e sidades, Spain, o he equali ica ion o he Spanish uni e si y sys em, and inanced by he Eu opean Union (Nex Gene a ionEU). We a e g a e ul o Elbland Bio-Muhle GmbH, especially Sebas ian S ein, o ui ul discussions and au hen ic lou samples and Sig id Benemann (BAM’s Su ace Analysis and In e acial Chemis y Di ision) o SEM measu emen s. The pa icles epo ed he ein a e a ailable upon eques o esea ch pu poses. ■REFERENCES (1) Johnson, R. L. Flow Cy ome y: F om Resea ch o Clinical Labo a o y Applica ions. Clin. Lab. Med. 1993,13 (4), 831−852. (2) Lambe , C. Flow Cy ome y, a Unique Bio echnology in Medical Applica ions. J. 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