Click and detect: Versatile ampicillin aptasensor enabled by click chemistry on a graphene-alkyne derivative

2207216 (1 o 10) © 2023 The Au ho s. Small published by Wiley-VCH GmbH www.small-jou nal.com Click and De ec : Ve sa ile Ampicillin Ap asenso Enabled by Click Chemis y on a G aphene–Alkyne De i a i e José M. R. Flauzino, Ma in-Alex Nalepa, Deme ios D. Ch onopoulos, Ve onika Šedajo á, Da id Panáček, Pe Jakubec, Pe a Küh o á, Ma in Pykal, Pa el Banáš, Aleš Panáček, A is ides Bakand i sos, and Michal O yepka* DOI: 10.1002/smll.202207216 he indisc imina e exposu e o mic o- o ganisms o an ibio ics. This equi es con olling an ibio ic use and b oad moni- o ing o hei le els in complex samples, such as ood, ap and was ewa e , u ine, and blood.[3] Ch oma og aphy and he enzyme-linked immunoso ben assay a e conside ed he gold s anda d me hods o an ibio ic de ec ion. Howe e , hey a e ime-consuming and equi e expensi e ins umen a ion and ained pe sonnel.[4] Thus, he de elopmen o poin -o -in e es an ibio ic de ec ion me hods ha a e as , cheap, e ec i e, sensi i e, and do no equi e sophis ica ed labo a o y equip- men o expe pe sonnel is c ucial. To his end, p ope ly designed nano-biosenso s mee all he abo e equi emen s, com- bining selec i e and apid bio ecogni ion o a ge analy es wi h a concen a ion- dependen signal esponse o quan i a i e ead-ou .[5] The mos e ec i e biosenso s ely on selec i e ecogni ion o an analy e in a com- plex sample ia speci ic binding on a high-a ini y bio ecep o , which adi ionally comp ises an enzyme o an ibody immobi- lized on an app op ia e suppo . Howe e , owing o he high cos and di icul handling o such delica e biomolecules, syn- he ic nucleic acids, such as ap ame s, may be be e al e na i es because hey a e mo e s able and simple o syn hesize.[6] An ap ame ’s nucleo ide sequence can be ailo ed o selec i ely bind Tackling he cu en p oblem o an imic obial esis ance (AMR) equi es as , inexpensi e, and e ec i e me hods o con olling and de ec ing an ibio ics in di e se samples a he poin o in e es . Cos -e ec i e, disposable, poin -o - ca e elec ochemical biosenso s a e a pa icula ly a ac i e op ion. Howe e , he e is a need o conduc i e and e sa ile ca bon-based ma e ials and inks ha enable e ec i e bioconjuga ion unde mild condi ions o he de elop- men o obus , sensi i e, and selec i e de ices. This wo k desc ibes a simple and as me hodology o cons uc an ap asenso based on a no el g aphene de i a i e equipped wi h alkyne g oups p epa ed ia luo og aphene chem- is y. Using click chemis y, an ap ame is immobilized and used as a suc- cess ul pla o m o he selec i e de e mina ion o ampicillin in eal samples in he p esence o in e e ing molecules. The elec ochemical ap asenso displayed a de ec ion limi o 1.36nM, high selec i i y among o he an ibi- o ics, he s o age s abili y o 4 weeks, and is e ec i e in eal samples. Addi- ionally, s uc u al and docking simula ions o he ap ame shed ligh on he ampicillin binding mechanism. The e sa ili y o his pla o m opens up wide possibili ies o cons uc ing a new class o ap asenso based on disposable sc een-p in ed ca bon elec odes usable in poin -o -ca e de ices. ReseaRch aR icle J. M. R. Flauzino, M.-A. Nalepa, D. D. Ch onopoulos, V. Šedajo á, D. Panáček, P. Jakubec, P. Küh o á, M. Pykal, P. Banáš, A. Bakand i sos, M. O yepka Regional Cen e o Ad anced Technologies and Ma e ials Czech Ad anced Technology and Resea ch Ins i u e (CATRIN) Palacký Uni e si y Olomouc Šlech i elů 27, Olomouc 783 71, Czech Republic E-mail: [email p o ec ed] The ORCID iden i ica ion numbe (s) o he au ho (s) o his a icle can be ound unde h ps://doi.o g/10.1002/smll.202207216. © 2023 The Au ho s. Small published by Wiley-VCH GmbH. This is an open access a icle unde he e ms o he C ea i e Commons A ibu- ion License, which pe mi s use, dis ibu ion and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. A. Panáček Depa men o Physical Chemis y Facul y o Science Palacký Uni e si y Olomouc 771 46, Czech Republic A. Bakand i sos Nano echnology Cen e Cen e o Ene gy and En i onmen al Technologies VSB – Technical Uni e si y o Os a a 17. lis opadu 2172/15 708 00, Os a a-Po uba, Czech Republic M. O yepka IT4Inno a ions VSB – Technical Uni e si y o Os a a 17. lis opadu 2172/15, Os a a-Po uba 708 00, Czech Republic 1. In oduc ion AMR de elopmen is becoming one o he mos se e e h ea s o public heal h his cen u y. Wo ldwide, 4.95 million dea hs we e associa ed wi h AMR in 2019,[1,2] and he dea h oll is expec ed o ise o mo e han 10 million by 2050.[2] One o he key measu es o es ic he de elopmen o AMR is o p e en Small 2023, 2207216 2207216 (2 o 10) www.ad ancedsciencenews.com © 2023 The Au ho s. Small published by Wiley-VCH GmbH www.small-jou nal.com a a ge using he sys ema ic e olu ion o ligands by exponen ial en ichmen (SELEX) echnique.[7] Cu en ly, many ap asenso s ely on complica ed de ec ion me hods based on nanopa icle agg ega ion o equi e a ge ampli ica ion.[8,9] Elec ochemical ap asenso s can simpli y he de ec ion o a ge molecules based on a cu en esponse ela ed o a con o ma ional change in he ap ame , which is conjuga ed o a edox p obe, a e a ge mol- ecule binding.[10] Howe e , hei use is so a limi ed o gold and glassy ca bon elec odes,[11] and low-cos , p in ed elec odes ha e no ye been explo ed due o hei nonuni o m su aces. Thus, new ca bon-based ansduce s a e highly desi ed. G aphene ul ills many equi emen s o an e ec i e ans- duce due o i s high conduc i i y, high su ace a ea, and low cos . Howe e , he low chemical eac i i y and hyd ophobic na u e o p is ine g aphene hampe s i s unc ionaliza ion[12,13] and a achmen o bio ecogni ion uni s,[14,15] as well as i s e ec i e handling and in eg a ion in de ices. The low densi y o elec ochemically ac i e de ec s in p is ine g aphene also limi s i s pe o mance in elec ochemical sensing,[16] while in oducing de ec s and unc ionali ies ia oxida ion owa d g aphene oxide (GO) u ns g aphene in o an insula o [17] and o en equi es gold co-deposi ion o inc ease he conduc i i y.[16] Reduc ion s eps can eco e pa o i s conduc i i y bu a he expense o diminishing he densi y o chemical unc ionali ies. Ano he limi a ion in using GO s ems om he la ge di e si y o oxygen unc ionali ies (e.g., hyd oxyl, ca boxyl, e he , epoxy, and ke o g oups),[18] making i s ep oducible, e ec i e and selec i e unc ionaliza ion e y challenging. The chemis y o luo og aphene[20] bypasses such sho com- ings by p o iding a la ge po olio o selec i ely and densely su ace- unc ionalized conduc i e g aphene de i a i es.[21–23] G aphene acid (GA),[24] wi h i s high dispe sibili y in aqueous media, la ge numbe o su ace-exposed ca boxylic g oups, elec ical conduc i i y, and biocompa ibili y, can be consid- e ed an ideal sca old o he ab ica ion o biosenso s. GA has al eady been used o elec ochemical sensing H2O2,[25] and i s ca boxylic g oups ha e been employed o co alen conjuga ion o biomolecules, such as enzymes.[26] Recen ly, i was also u i- lized o conjuga ion wi h an DNA p obe speci ic o he pig mi ochond ial genome o p epa e a label- ee elec ochemical genosenso .[27] The conjuga ion elied on pep ide bond o ma- ion be ween ca boxyl g oups o GA and amines o he bio- molecule ia ca bodiimide chemis y. Howe e , conjuga ion o ap ame s equi es speci ic chemis y conjuga ion me hods o a oid ap ame damage, especially app oaches ha do no ely on amino g oups because o hei abundance in biomolecules and abili y o o m nonspeci ic c osslinks. In his espec , click chemis y ep esen s an ideal bioconju- ga ion me hod. The e m was coined by he ecen Nobel P ize awa dee K. Ba y Sha pless in 2001 o desc ibe eac ions wi h high yields, wide scope, and no o ew byp oduc s.[27] One clas- sical click eac ion is coppe -ca alyzed azide-alkyne cycloaddi- ion (CuAAC), in which an azide eac s wi h an e minal alkyne o o m a i e-membe ed iazole ing unde ambien condi- ions.[28] Howe e , he eac ions condi ions mus be well- uned, as i s success depends on innume ous ac o s such as sol en , ca alys concen a ion, and numbe o ac i e si es and he use o biomolecules equi es bu e solu ions and speci ic condi- ions. The use o CuAAC o cons uc ap asenso s is s ill a an ea ly s age,[29,30] and i s po en ial on g aphene subs a es has no ye been explo ed. In his wo k, we p esen a new alkyne- e mina ed g aphene de i a i e (GA-NH-YN, Figu e 1) capable o unde going acile conjuga ion wi h bio ecogni ion uni s ia click chemis y. We demons a e s aigh o wa d CuAAC conjuga ion wi h a edox- p obe-modi ied DNA ap ame ha selec i ely binds ampicillin. The de eloped elec ochemical ap asenso displayed high selec- i i y and sensi i i y o ampicillin e en in complex samples, such as ap wa e , sali a, and milk. The de eloped g aphene de i a i e p o ides a click-chemis y- eady sys em o conjuga- ion wi h comme cially a ailable azide-modi ied bio ecogni ion uni s as a e sa ile pla o m o he design o a wide po olio o biosenso s. Fu he mo e, GA-NH-YN allows he ab ica ion o lexible p in ed elec odes on disposable subs a es o use as low-cos , poin -o -in e es elec ochemical senso s ha can be ope a ed ia a mobile phone. Impo an ly, hese ea u es a e combined wi h high selec i i y, e en in p esence o s uc- u ally simila an ibio ics, and long shel -li e, while o e ing a de ec ion limi o ampicillin ha is eigh - old lowe han he maximum esidue limi o ampicillin in milk. Small 2023, 2207216 Figu e 1. A) Reac ion scheme depic ing click chemis y be ween GA-NH-YN and a DNA ap ame bea ing an azide moie y. B) FTIR spec a, and C) p o- posed s uc u es o he s a ing nanoma e ial (g aphene acid – GA), a e conjuga ion wi h p opa gylamine (GA-NH-YN) and click-chemis y eac ion wi h he ap ame (GA-T iazole-DNA). D) Elemen al composi- ions o species in ol ed in he click-chemis y eac ion by XPS analysis (see Figu e S1, Suppo ing In o ma ion o su ey XPS spec a). 16136829, 0, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/smll.202207216 by Technical Uni e si y Os a a, Wiley Online Lib a y on [28/11/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License 2207216 (3 o 10) www.ad ancedsciencenews.com © 2023 The Au ho s. Small published by Wiley-VCH GmbH www.small-jou nal.com 2. Resul s and Discussion 2.1. Nanoma e ial Syn hesis and Cha ac e iza ion The ca boxylic g oups o GA we e eac ed wi h he p ima y amine uni s o p opa gylamine ia ca bodiimide chemis y, yielding he co esponding alkyne- e mina ed g aphene de i a- i e (GA-NH-YN, Figu e 1). Subsequen ly, GA-NH-YN was conjuga ed ia click chemis y o he DNA ap ame bea ing an azide moie y a e he o ma ion o a 5-membe ed iazole ing (GA-T iazole-DNA) ia 1,3-dipola CuAAC.[29] Chemical changes du ing his de i a iza ion o GA we e moni o ed by Fou ie - ans o m in a ed (FTIR) spec oscopy (Figu e1B). The FTIR spec um o GA-NH-YN displayed a peak a 2190 cm−1, asc ibed o he e minal alkyne g oup, con i ming he success ul coupling o GA wi h p opa gylamine. Mo eo e , he in ensi y o he ca boxyl g oup band in he s a ing GA, cen e ed a 1720 cm−1, was signi ican ly educed in compa ison o he o he GA bands due o amide bond o ma ion.[24] A e he click eac ion o he alkyne g aphene de i a i e wi h he azide-bea ing ap ame , he band o he alkyne g oup anished because o i s con e sion o a 1,2,3- iazole ing.[32] The suc- cess ul co alen conjuga ion o he DNA ap ame on o GA-NH- YN was indica ed by he appea ance o h ee sha p bands a 3400, 3360, and 3200 cm−1 a ibu able o N-H s e ching modes o he guanine bases o he ap ame .[33] X- ay pho oelec on spec oscopy (XPS) u he p o ed he ap ame ’s success ul a achmen on GA-NH-YN owing o he eme gence o a high a omic phospho us con en (0.7 a .%, Figu e1D, Figu e S1, Table S1, Suppo ing In o ma ion) in he GA-T iazole-DNA p oduc . In compa ison, he p oduc ob ained by simple physical mixing o GA-NH-YN and he ap ame (wi hou coppe and he asco ba e ca alys ) showed lowe P con- en due o nonspeci ic so p ion, e i ying indi ec ly co alen g a ing o he DNA ap ame in he GA-T iazole-DNA p oduc . In o ma ion abou he mo phology o GA-T iazole-DNA was collec ed by high- esolu ion ansmission elec on mic oscopy (HR-TEM), ene gy dispe si e spec oscopy (EDS) elemen al mapping, a omic o ce mic oscopy (AFM), and classical mole- cula dynamics (MD) simula ions (Figu e 2). A ep esen a i e HR-TEM image showed ha GA-T iazole-DNA consis ed o e y ew laye s o g aphene (Figu e2A). EDS elemen al map- ping con i med he p esence and homogeneous dis ibu ion o he DNA ap ame ia de ec ion o phospho ous in he sample (Figu e2F). Acco ding o AFM measu emen s, he hicknesses o GA-NH-YN and GA-T iazole-DNA we e 1.6nm (o 3.1nm o double-laye s uc u e) and 1.9nm, espec i ely (Figu e S4, Sup- po ing In o ma ion and Figu e2G). The heigh o GA-NH-YN de i ed om MD simula ions was 1.7 ± 0.01nm, co esponding well o he AFM da a. MD calcula ions o GA-T iazole-DNA ca - ied ou in wa e sugges ed ha he DNA co alen ly ancho ed o he alkyne- e mina ed g aphene occu ed in ei he a hai pin- like o iplex s uc u e (Figu e 2H, see also Me hodology sec ion and empe a u e eplica-exchange MD simula ions (T-REMD) discussed la e o u he de ails). The simula ions indica ed ha he heigh o GA-T iazole-DNA in wa e eached 3.6 ± 0.2 and 4.8 ± 0.3nm o he hai pin-like and iplex old s uc u es, espec i ely. These alues a e highe han hose ob ained by AFM, pe haps because in he AFM expe imen , sample d ying caused he DNA ap ame o lose i s na i e old and s ack mo e on he g aphene su ace. UV-Vis spec oscopy (Figu e S6, Suppo ing In o ma ion) also sugges ed DNA a achmen o GA-NH-YN based on an inc ease in he signal a ≈260 nm a e he click-chemis y eac ion. Ze a po en ial measu emen s (Table S2, Suppo ing In o ma ion) e ealed a highe nega i e po en ial o GA- T iazole-DNA (−20.9 mV) in compa ison wi h ba e GA-NH- YN (−10.7 mV) and he con ol sample ob ained om simple mixing wi h DNA wi hou ca alys (−13.8mV), indica ing ha he nega i ely cha ged DNA ap ame was success ully a ached o he nanoma e ial. The Raman spec a (Figu e S7, Suppo ing In o ma ion) show dis inc i e D band (a ≈1330 cm−1) and G band (≈1595 cm−1) bands, bo h e lec ing he sp2 hyb idized ca bon (D band wi h nea by sp3 hyb idized ca bon, ypically a de ec o acancy and G band a oma ic sp2 a ea).[33,34] The ID/IG a io o all h ee samples emained almos unchanged, wi h he alue anging only 1.28–1.24. Howe e , sha p bands ≈2800 cm−1 o he GA-T iazole-DNA sample can be asc ibed o he p es- ence o he C–H ib a ions coming om he alipha ic linke s o he conjuga ed DNA.[35,36] 2.2. Biosenso Design and Op imiza ion To ine- une he click-chemis y eac ion o op imize he elec- ochemical esponse, CuAAC eac ion pa ame e s need o be s udied on o he solid su ace o he elec ode. The a io be ween coppe and educing agen and he eac ion ime a e key pa am- e e s and hus we e e alua ed in his wo k. A e 60min o eac- ion, he me hylene blue (MB) educ ion cu en peak ( he edox epo e on he DNA) emained cons an (Figu e 3A), indica ing ha he su ace was sa u a ed wi h DNA. Thus, a eac ion ime o 60min was selec ed o u he s eps. The op imum asco - ba e concen a ion was ound o be 20mM (Figu e3B): lowe concen a ions esul ed in low eac ion yields and elec ochem- ical signal, whe eas highe concen a ions appea ed o hinde he eac ion o e en damage he ap ame .[38] De ec ion o ampicillin by squa e wa e ol amme y (SWV) was mo e e icien a a high equency (100Hz) and low ampli- ude (10 mV), esul ing in a signal inc ease o 90% in com- pa ison o ha o he blank, i.e., in he absence o ampicillin (Figu e3C,D). Thus, he ap asenso exhibi ed “signal-on” ype beha io due o he cu en esponse inc ease upon a ge binding owing o con o ma ional changes o he ap ame and consequen app oxima ion o he edox p obe on he elec ode/ g aphene su ace. A low equencies (25 Hz), we obse ed he opposi e beha io , as he signal gain was nega i e, i.e., he esponse o he a ge was lowe han in he blank, indica ing a “signal-o ” esponse. P e ious s udies ha e indica ed ha such beha io is dependen on he equency applied du ing SWV.[38] In ou wo k, a equency o 100 Hz was chosen o he nex s eps due o he highe signal gain. Elec ochemical expe imen s wi h he [Fe(CN)6]3-/4− edox p obe p o ided aluable in o ma ion abou he es ed ma e ials and con i med success ul ap ame binding. Cyclic ol amme y (CV) (Figu e3E) demons a ed ha he elec ode modi ied wi h alkyne-g aphene had signi ican ly imp o ed elec ochemical pe o mance since i showed highe cu en signals compa ed Small 2023, 2207216 16136829, 0, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/smll.202207216 by Technical Uni e si y Os a a, Wiley Online Lib a y on [28/11/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License 2207216 (4 o 10) www.ad ancedsciencenews.com © 2023 The Au ho s. Small published by Wiley-VCH GmbH www.small-jou nal.com o ba e, p econdi ioned, and ap ame -modi ied elec odes. Elec- ochemical impedance spec oscopy (EIS) analysis and i ing wi h an equi alen Randles ci cui (Figu e3F) e ealed ha he ba e sc een-p in ed ca bon elec ode (SPCE) was e y esis i e wi h a high cha ge- ans e esis ance (RCT) o 3620 ± 145 Ω, bu he acidic p econdi ioning imp o ed he conduc i i y, low- e ing RCT o 1640 ± 90 Ω. Modi ica ion o he SPCE wi h he nanoma e ial lowe ed his alue u he (814 ± 58 Ω), high- ligh ing he conduc i e p ope ies o he g aphene de i a i e. A e he click-chemis y eac ion wi h he ap ame , he e was an inc ease in RCT (1420 ± 72 Ω). This may be due o he ne nega i e cha ge o DNA, which epels he nega i ely cha ged [Fe(CN)6]3-/4− ions, combined wi h g ea e s e ic hind ance a he elec ode su ace. 2.3. Ampicillin De ec ion The cu en esponse o he ap asenso a ied depending on he concen a ion o ampicillin (Figu e 4A). As he ap ame became sa u a ed wi h he a ge , highe concen a ions showed a simila esponse, esul ing in a loga i hmic-shaped cu e (Figu e S8, Suppo ing In o ma ion) ha could be line- a ized by applying a log unc ion o yield he ollowing equa ion ip= (2.2±0.1) * log cAMP+ (6.8 ±0.6) ( = 0.991, n= 21), whe e ip is he peak cu en o MB educ ion (in µA) and cAMP is he ampicillin concen a ion (in nM, see Figu e 4B). The limi o de ec ion was calcula ed as 3.3*σ/S, whe e σ is he s anda d de ia ion o he blank and S is he slope o he cu e, esul ing in 1.36 nM (o ≈0.5 µg L−1). This limi is highe han ha o o he epo ed biosenso s o ampicillin (Table S3, Suppo ing In o ma ion). Howe e , i is eigh - old lowe han he Eu opean maximum esidue limi s in milk (4µg L−1),[40,41] sa is ying he egula o y and ma ke needs. Mo eo e , he p esen ed pla o m is he only one epo ed o da e ha is based on disposable ca bon elec odes, o e ing supe io cos bene i s, and hus a highly compe i i e solu ion o ex ensi e sample sc eening. To e alua e he ap asenso selec i i y, ou o he an ibio ics we e es ed. The ap asenso has an inhe en esponse (back- g ound cu en ) o 10µA, which co esponds o he me hylene blue educ ion cu en in bu e solu ion wi hou any binding molecule in he ap ame . The binding wi h he speci ic a ge Small 2023, 2207216 Figu e 2. HR-TEM/EDS o he GA-T iazole-DNA: A) b igh ield, B) da k ield, C) o e lap o he elemen al mapping o D) ca bon, E) ni ogen, and F) phospho ous a oms. G) AFM analysis o GA-T iazole-DNA. H) Snapsho s om ee 100ns long MD simula ions showing he iplex and hai pin loop s uc u e o he ancho ed DNA-ap ame on g aphene acid. Bo h mo i s showed good s uc u al compa ibili y wi h he subs a e. The me hylene blue molecule (connec ed o he 5’ end o he ap ame ) is shown in cyan. Wa e molecules and ions a e omi ed o cla i y. 16136829, 0, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/smll.202207216 by Technical Uni e si y Os a a, Wiley Online Lib a y on [28/11/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License 2207216 (5 o 10) www.ad ancedsciencenews.com © 2023 The Au ho s. Small published by Wiley-VCH GmbH www.small-jou nal.com (ampicillin) makes he cu en esponse double due o con o - ma ional changes. As he esponse o he ap asenso in con- ac wi h o he an ibio ics, including wo s uc u al analogs penicillin G and amoxicillin, was he same as he absence o a ge (blank), he ap asenso is highly speci ic (Figu e4C). The esponse in he p esence o azi h omycin and e acycline was also equi alen o ha in he blank solu ion (PBS-MgCl2). A e 4 weeks o s o age, he ap asenso cu en esponse o ampi- cillin inc eased by 5%, p obably due o changes in he con o - ma ion o he ap ame , in which he edox p obe can be close o he elec ode su ace (Figu e4D). Ne e heless, his indica es excellen s abili y o e ime and demons a es i s po en ial as a p ac ical and long shel -li e p oduc . Nex , we e alua ed he po able se up based on a mic opo- en ios a connec ed o a sma phone (Figu e 5A) o poin -o - ca e analysis and ound ha i ga e only a small shi in peak po en ial (Figu e5B), which did no a ec he senso ’s pe o - mance. Fu he , he analysis in eal samples demons a ed he abili y o he ap asenso o selec i e de ec ion e en in complex media. The cu en esponse o he senso in eal samples Small 2023, 2207216 Figu e 3. Click-chemis y in-si u op imiza ion. Peak cu en o me hylene blue educ ion as a unc ion o A) ime, and B) asco ba e concen a ion. C) Pe cen age signal gain o he a ge and blank o di e en SWV pa ame e s. D) Squa e wa e ol ammog ams o me hylene blue educ ion wi h op imized pa ame e s (100Hz, 10mV) wi h/wi hou ampicillin and in he absence o ap ame . E) Cyclic ol ammog ams, and F) impedance spec a we e eco ded a a ious s ages o he biosenso cons uc ion wi h edox p obe (5mM [Fe(CN)6]3-/4− in PBS solu ion 10mM, pH = 7.4). All po en ials a e e sus me allic sil e . 16136829, 0, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/smll.202207216 by Technical Uni e si y Os a a, Wiley Online Lib a y on [28/11/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License 2207216 (6 o 10) www.ad ancedsciencenews.com © 2023 The Au ho s. Small published by Wiley-VCH GmbH www.small-jou nal.com spiked wi h ampicillin was close o he signal eco ded in he bu e (Figu e5C). Spiked ap wa e showed a eco e y a e o 104%, while sali a and milk had 90% and 88% eco e y a es, espec i ely, e en hough hey a e highly complex samples con- aining many po en ial in e e ing species, such as p o eins, ca bohyd a es, and lipids (Table 1). Such supe b pe o mance and ope a ion wi h he mic opo en ios a connec ed o a sma - phone opens he possibili y o using he se up o eal-li e and a o dable poin -o -ca e analysis. 2.4. Mechanism o Ac ion To da e, he 3D s uc u e o he apo- and holo- o ms o he ampicillin-binding DNA ap ame and he con o ma ion o he complex emain unknown. To gain some p elimina y insigh s in o he mechanism o he ampicillin-binding DNA ap ame , we used compu a ional chemis y ools ha can desc ibe he biomolecula sys ems wi h unp eceden ed empo al and spa ial esolu ion.[42,43] We used se e al se s o classical and Small 2023, 2207216 Figu e 4. Ampicillin de ec ion. A) Rep esen a i e ol ammog ams o he esponse o he ap asenso in he p esence o di e en ampicillin concen a- ions. B) Calib a ion cu e o he ap asenso . C) Nega i e con ol es s, wi h he s uc u es o he ampicillin analogues. D) S abili y o e ime s udy. All po en ials a e e sus me allic sil e . Figu e 5. Real sample de ec ion. A) Se up comp ises a sma phone connec ed o a mic opo en ios a holding a sc een-p in ed ca bon elec ode. B) Vol ammog ams we e eco ded in di e en samples. C) Ba cha o he cu en esponse in di e en samples. 16136829, 0, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/smll.202207216 by Technical Uni e si y Os a a, Wiley Online Lib a y on [28/11/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License 2207216 (7 o 10) www.ad ancedsciencenews.com © 2023 The Au ho s. Small published by Wiley-VCH GmbH www.small-jou nal.com enhanced-sampling all-a om MD simula ions o s udy he con o ma ional beha io o DNA ap ame (see Table S4, Sup- po ing In o ma ion o a comple e lis o simula ions). We pe o med T-REMD simula ions s a ing om he un olded s a e o he DNA single-s anded ap ame in he p esence o absence o ampicillin. Al hough he simula ions we e no well con e ged, hey p o ided aluable insigh s in o he s uc u al p e e ences o he ap ame sequence and binding con o ma- ion o ampicillin o he DNA nucleo ides. Clus e ing and base pai ing analyses (Figu e S9–11, Suppo ing In o ma ion) iden- i ied he loca ions whe e he sys em ended o o m s ems, and con e sely, in combina ion wi h he s acking analysis, hey iden i ied posi ions in he sequence ha we e p one o bending (Figu e S12–14, Suppo ing In o ma ion) and o ma ion o loops. All T-REMD simula ions, wi h o wi hou ampicillin, showed ha he ap ame had a endency o o m wo loops posi ioned a ou dis inc si es in he sequence (Figu es S9 and S10, Suppo ing In o ma ion), he eby o ming a iplex-like s uc u e. In he T-REMD simula ions wi h ampicillin, we ocused on he beha io o he an ibio ic i sel , i.e., how and whe e i is bound. The simula ions sugges ed ha he DNA bound o ampicillin in i s ze o-ne -cha ge zwi e ionic o m. The e was almos no ampicillin-DNA binding in he T-REMD simula ions o dep o ona ed nega i ely cha ged ampicillin (wi h a neu al N18 amino g oup). In he zwi e ionic o m, ampicillin bound o DNA nucleo ides p edominan ly ia he N18 ammonium g oup (Figu e 6). This g oup s ongly a o ed binding o hy- mine nucleo ides, and showed modes binding o guanine and cy osine and no endency o in e ac wi h adenine. In addi- ion, we obse ed ha he R3 phenyl ing o ampicillin ended o s ack o he nucleobases. This migh sugges ha hyd ogen bonding o he N18 ammonium g oup oge he wi h s acking o he R3 phenyl ing a e in ol ed in he ecogni ion o ampi- cillin by he ap ame , which would explain he selec i i y o he ap ame o ampicillin o e penicillin G. Howe e , he sam- pling o ampicillin binding was oo limi ed o be su icien ly p edic i e. 3. Conclusions We syn hesized a new conduc i e g aphene de i a i e bea ing alkyne g oups ha can unde go click-chemis y eac ion wi h biomolecules unde mild condi ions. Using he clas- sical CuAAC click eac ion, we de eloped a simple, as , and s aigh o wa d p o ocol o ap ame conjuga ion on o dispos- able, sc een-p in ed elec odes ha had been p emodi ied wi h he alkyne-g aphene de i a i e. The de eloped ap asenso was shown o be sensi i e and selec i e, as demons a ed o he case o he an ibio ic ampicillin. The design o he pla o m is e sa ile, as he p o ocol can be used wi h di e en ap ame s o de ec a wide a ie y o a ge analy es, hus enabling low-cos and disposable sc een-p in ed ca bon elec odes o be used o he cons uc ion o sensi i e, selec i e, and s able poin -o -ca e elec ochemical senso s. 4. Expe imen al Sec ion Ma e ials: All eagen s we e used as ecei ed wi hou u he pu i ica ion. P opa gylamine, anhyd ous e ahyd o u an (THF), and N-(3- dime hylaminop opyl)-N′-e hylca bodiimide hyd ochlo ide (EDC-HCl) we e pu chased om Sigma-Ald ich. E hyl isoni osocyanoace a e (Oxyma) was pu chased om Al a Aesa . Ampicillin, amoxicillin, penicillin G, azi h omycin, e acycline, phospha e bu e ed saline (PBS 10 mM, pH = 7.4), po assium e ocyanide, po assium e icyanide, magnesium chlo ide, coppe sul a e, sodium asco ba e, and sul u ic acid we e pu chased om Sigma-Ald ich/Me ck. Deionized wa e (ρ= 18.2 MΩ cm−1) was used o p epa e all solu ions. The ap ame o ampicillin was selec ed by Song and collabo a o s,[44] which pe o med op ical es s and chose AMP17 sequence as he bes op ion o binding o ampicillin. I was pu chased om Biome s, Ge many, wi h modi ica ions a bo h ends (Figu e S2, Suppo ing In o ma ion) Small 2023, 2207216 Table 1. The eco e y a e o he ap asenso in spiked eal samples. Sample cAMP spiked cAMP measu ed Reco e y a e Tap wa e 1 mM 1.040 mM 104% 50% Sali a 1 mM 0.904 mM 90.4% 50% Milk 1 mM 0.882 mM 88.2% Figu e 6. The popula ion o H-bonds in e ac ions and s acking in e ac ion be ween ampicillin and DNA ap ame in he e e ence 298 K eplica calcu- la ed om he las mic osecond o T-REMD simula ion o holo o m (zwi e ionic elec os a ically neu al AMP(0)) o he d(CGGGCGGTTGTATAGCGG) sequence. The blue boxes ep esen H-bonds be ween ampicillin and he backbone, g een boxes be ween ampicillin and DNA bases, and o ange boxes ep esen s acking in e ac ions be ween phenyl ing o ampicillin and DNA bases. 16136829, 0, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/smll.202207216 by Technical Uni e si y Os a a, Wiley Online Lib a y on [28/11/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License 2207216 (8 o 10) www.ad ancedsciencenews.com © 2023 The Au ho s. Small published by Wiley-VCH GmbH www.small-jou nal.com Small 2023, 2207216 as ollows: 5′-(me hylene blue)-GCGGGCGGTTGTATAGCGG-(azido- p oline)-3’. The ap ame was esuspended in deionized wa e a 1mM. Fo op imal binding, he ap ame was olded in o i s e ia y s uc u e. P io o use, an aliquo o 10µL he ap ame solu ion was dilu ed o 0.1mM in PBS con aining 2mM MgCl2 (PBS-MgCl2), and hen hea ed o 5min a 70 °C and cooled o oom empe a u e (RT). Nanoma e ial Syn hesis: GA was syn hesized acco ding o a p e iously published epo .[24] Fo he conjuga ion o GA wi h p opa gylamine o o m he GA-NH-YN, a suspension o GA (30 mg) was p epa ed in anhyd ous THF (10 mL) in a h ee-neck ound-bo om glass lask, hen EDC-HCl (444mg, 2.32mmol) and e hyl isoni osocyanoace a e (330mg, 2.32mmol) we e added and he esul ing mix u e was s i ed o 30 min a RT unde a ni ogen a mosphe e. A e wa d, 440 µL (6.87mmol) o p opa gylamine was added d opwise o he suspension and he eac ion mix u e was s i ed o 120h a RT. Nex , he mix u e was dilu ed in e hanol and he p ecipi a e was collec ed by cen i uga ion. The inal p oduc was isola ed by consecu i e cen i ugal washing s eps using N,N-dime hyl o mamide, wa e , e hanol, and dichlo ome hane. Click Chemis y in Solu ion: Fo FTIR-ATR, XPS, SEM-EDS, TEM, AFM, ze a po en ial measu emen s, and UV- is and Raman spec oscopy analysis, he click-chemis y eac ion be ween GA-NH-YN and he ap ame was pe o med in solu ion as ollows: 700 µL o a 1mg mL−1 suspension o GA-NH-YN was mixed wi h 100 µL o 0.1 mM azide- ap ame solu ion, 100 µL o 1 mM CuSO4 solu ion and 100 µL o 3 mM asco ba e solu ion. The eac ion was le o p oceed o 48 h unde agi a ion (1000 pm) and a oom empe a u e (25 °C). To a oid damaging he biomolecule, sonica ion was no used. The nanoma e ial was pu i ied by cen i uga ion a 14 000 pm o 10 min and hen esuspended in 500 µL deionized wa e . This cen i ugal washing was epea ed wice mo e. GA-NH-YN physical mix u es wi h only ap ame o coppe and asco ba e we e used as con ols. Biosenso Cons uc ion (in si u Click Chemis y): Comme cially a ailable SPCE we e used as a disposable sensing pla o m (DRP-110, Me ohm D op Sens, Spain). They comp ised a ca bon disk wo king elec ode (4mm diame e ), ca bon auxilia y elec ode, and sil e pseudo- e e ence elec ode. Be o e use, p econdi ioning was ca ied ou in 0.1M H2SO4 solu ion by CV (−1.5–+1.5 V, 5 cycles, 100 mV s−1) o emo e o ganic c osslinks om he ca bon ink, inc easing he SPCE unc ionali y and ep oducibili y. An Au olab po en ios a (model PGSTAT128N) was used o all elec ochemical analyses unless o he wise speci ied. The alkyne-g aphene was deposi ed on o he wo king elec ode by d op-cas ing acco ding o a p e iously es ablished s a egy,[27] in which he nanoma e ial suspension was cen i uged o 10min a 10 000 pm and 20 µL o he supe na an was deposi ed on o he wo king elec ode su ace. A e d ying a oom empe a u e, he CuAAC eac ion was pe o med in si u a oom empe a u e (25 °C). A 100µM solu ion o he ap ame was mixed wi h 1mM CuSO4 and a ious concen a ions o sodium asco ba e solu ion in a 2:1:1 p opo ion, espec i ely. Di e en sodium asco ba e concen a ions (3, 5, 10, 20, 30, 40, and 50mM) and eac ion imes (15, 30, 45, 60, and 90min) we e es ed o op imize he eac ion. A e wa d, he elec ode su ace was washed wi h PBS-MgCl2. Immobilized ap ame was de ec ed by moni o ing he educ ion peak o MB by SWV using di e en equencies (10, 25, 50, and 100 Hz) and ampli udes (10, 25, 50, and 100mV) in PBS-MgCl2. All measu emen s we e made in iplica e and p esen ed as mean ± s anda d de ia ion. Elec ochemical s udies o he biosenso cons uc ion s eps we e pe o med using 5 mM [Fe(CN)6]3-/4- edox p obe in PBS solu ion (10mM, pH = 7.4). CV was measu ed be ween −0.4V and + 0.7V a scan a e o 50mV s−1, EIS was measu ed a E1/2= 0.115V wi h a equency ange o 0.1Hz o 10kHz and ampli ude o 10mV. Sample De ec ion: PBS-MgCl2. con aining di e en ampicillin concen a ions (10, 1, 100, 10, 1, 100, and 10nM) was added d opwise o he su ace o he modi ied wo king elec ode and le o incuba e o 30min. SWV was pe o med o obse e he MB peak (po en ial ange: 0–−0.6V, equency: 100Hz, ampli ude: 10mV). Solu ions o PBS-MgCl2 con aining di e en an ibio ics (amoxicillin, penicillin G, azi h omycin, e acycline) a 1mM concen a ion we e used as nega i e con ols. The s abili y o he ap ame o e ime was e alua ed by keeping he di e en p epa ed elec odes a 4 °C and measu ing he esponse in a solu ion o 1mM ampicillin in PBS-MgCl2 in a pe iod o 4 weeks. Fo es eal sample de ec ion, ap wa e , 50% dilu ed eshly collec ed sali a, and 50% dilu ed milk (1.5% a ) spiked wi h 1mM ampicillin we e used and he eco e y pe cen age was calcula ed. A se up consis ing o a mic opo en ios a (Sensi Sma , Palm Sens) connec ed o a sma phone was u ilized. The baseline o all squa e wa e ol ammog ams was sub ac ed and he peak cu en was calcula ed using NOVA 2.1 and PST ace 5.9 so wa e. To aid he p esen a ion, he ol ammog ams we e in e ed and he mos ep esen a i e example was p esen ed. O igin 2018 so wa e was u ilized o plo all da a. Simula ion Se up: The un olded s a ing s uc u e o he DNA ap ame used in REMD olding and classical g aphene-g a ed simula ions wi h sequence d(GCGGGCGGTTGTATAGCGG) was p epa ed using he Nucleic Acid Builde ool in AMBER.[44] Addi ionally, o he g aphene-g a ed model, he seconda y s uc u e o he ap ame was p edic ed using he M old se e ,[45] con e ed in o a 3D s uc u e using he RNACompose se e ,[46] and ancho ed in he middle o he g aphi ic su ace. GA ( unc ionaliza ion deg ee 13%, o which 60% was addi ionally conjuga ed wi h he amino alkyne, and he emaining unmodi ied ca boxylic g oups we e simula ed in hei dep o ona ed o m) was modeled as a pe iodic shee in he xy plane wi h dimensions ≈99 × 101 Å, and un unc ionalized g aphi ic ca bons we e ea ed as uncha ged Lenna d-Jones sphe es. The pa ame e s o ampicillin in di e en p o ona ion s a es, MB and azido-p oline (N3-P o) we e adop ed om he GAFF o ce- ield. Pa ial cha ges o he alkyne- e mina ed GA (modeled on a small, unc ionalized py ene model), N3- P o linke s, MB and an ibio ics we e assessed using he RESP me hod a he HF/6-31G* le el o heo y[47] using he Gaussian 16 p og am.[48] Fo each se o simula ions (see Table SX1, Suppo ing In o ma ion), all ele an molecules we e subsequen ly assembled in o a single sys em and he AMBER18[49] o GROMACS 5.0 (in he case o a sys em wi h pe iodic g aphene)[50] packages we e used o gene a e he s a ing opology and coo dina es and un simula ions.T-REMD[51] and classical MD simula ions o he ap ame we e pe o med wi h he AMBER DNA o ce- ield OL21[52] including he pa mbsc0[53–55] o ce ield wi h χOL4[56]+ εζOL1[57]+ βOL1[58]+ α/γ[52] modi ica ions. The simula ions we e pe o med by combining he SPC/E[59] explici wa e model and ∼10mM KCl sal excess using Joung-Chea ham ion pa ame e s.[60] P io o he simula ions, he p epa ed sys ems we e minimized and equilib a ed using he s anda d equilib a ion p o ocol desc ibed in he Suppo ing In o ma ion. The inal p oduc ion un o he ap ame -g aphene sys em was ca ied ou in he NVT ensemble o 100ns. T-REMD simula ions we e un using 64 eplicas a empe a u es anging om 278 o 461 K. The 64 di e en con o ma ions we e chosen as s a ing s uc u es o ensu e be e con e gence. The simula ion ime o he T-REMD simula ions was a leas 4 µs (Table S4, Suppo ing In o ma ion). Suppo ing In o ma ion Suppo ing In o ma ion is a ailable om he Wiley Online Lib a y o om he au ho . Acknowledgemen s The au ho s g a e ully acknowledged suppo om he Technology Agency o he Czech Republic, P og am TREND (GEFSEM, FW01010183), and he ERDF/ESF p ojec “Nano4Fu u e” (No. CZ.02.1.01/0.0/0.0/16 _019/0000754). The au ho s acknowledged he assis ance p o ided by he Resea ch In as uc u e NanoEn iCz, suppo ed by he Minis y o Educa ion, You h and Spo s o he Czech Republic unde P ojec No. LM2018124. This wo k was suppo ed by he Minis y o Educa ion, You h and Spo s o he Czech Republic h ough he e-INFRA CZ (ID: 90140). This p ojec has ecei ed unding om he Eu opean 16136829, 0, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/smll.202207216 by Technical Uni e si y Os a a, Wiley Online Lib a y on [28/11/2023]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License 2207216 (9 o 10) www.ad ancedsciencenews.com © 2023 The Au ho s. Small published by Wiley-VCH GmbH www.small-jou nal.com Small 2023, 2207216 Union’s Ho izon Eu ope esea ch and inno a ion p og am unde g an ag eemen No 101059266. MO g a e ully acknowledged an ERC consolida o g an (683024) om Ho izon 2020. AB acknowledges unding om he Czech Science Founda ion (p ojec No 19–27454X). The au ho s hank Jiří Hošek, Ondřej Tomanec, and Klá a Čépe o conduc ing he SEM, AFM, and CPEM measu emen s. Con lic o In e es The au ho s decla e no con lic o in e es . Da a A ailabili y S a emen The da a ha suppo he indings o his s udy a e a ailable a ZENODO (h ps://doi.o g/10.5281/zenodo.7509720). Keywo ds an ibio ic de ec ion, ap ame s, biosenso s, g aphene acid, sc een p in ed ca bon elec odes Recei ed: No embe 20, 2022 Re ised: Decembe 20, 2022 Published online: [1] C. J.Mu ay, K. S.Iku a, F.Sha a a, L.Swe schinski, G. R.Aguila , A. G ay, C. Han, C. Bisignano, P. Rao, E. Wool, S. C. Johnson, A. J. B owne, M. G. Chipe a, F. Fell, S. Hacke , G. Haines- Woodhouse, B. H. K.Hamadani, E. A. P.Kuma an, B.McManigal, R. Aga wal, S. Akech, S. Albe son, J. Amuasi, J. And ews, A.A a kin, E.Ashley, F.Bailey, S.Bake , B.Basnya , A.Bekke , e al., Lance 2022, 399, 629. 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