A Novel NanoMIP-SPR Sensor for the Point-of-Care Diagnosis of Breast Cancer

Ci a ion: E ol, K.; Hasabnis, G.; Al in as, Z. A No el NanoMIP–SPR Senso o he Poin -o -Ca e Diagnosis o B eas Cance . Mic omachines 2023,14, 1086. h ps://doi.o g/10.3390/mi14051086 Academic Edi o s: Adil Denizli and Ye¸se en Saylan Recei ed: 24 Ap il 2023 Re ised: 15 May 2023 Accep ed: 18 May 2023 Published: 21 May 2023 Copy igh : © 2023 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). mic omachines A icle A No el NanoMIP–SPR Senso o he Poin -o -Ca e Diagnosis o B eas Cance Kadi E ol 1,2, Gau i Hasabnis 1and Zeynep Al in as 1,3,* 1Ins i u e o Ma e ials Science, Facul y o Enginee ing, Kiel Uni e si y, 24143 Kiel, Ge many; [email p o ec ed] (K.E.); [email p o ec ed] (G.H.) 2 En i onmen al Heal h P og am, Depa men o Medical Se ices and Techniques, Voca ional School o Heal h Se ices, Hi i Uni e si y, Co um 19030, Tu key 3Kiel Nano, Su ace and In e ace Science (KiNSIS), Kiel Uni e si y, 24118 Kiel, Ge many *Co espondence: [email p o ec ed]; Tel.: +49-(0)431-880-6198 Abs ac : Simple, as , selec i e, and eliable de ec ion o human epide mal g ow h ac o ecep o 2 (HER2 ) is o u mos impo ance in he ea ly diagnosis o b eas cance o p e en i s high p e alence and mo ali y. Molecula ly imp in ed polyme s (MIPs), also known as a i icial an ibodies, ha e ecen ly been used as a speci ic ool in cance diagnosis and he apy. In his s udy, a minia u ized su - ace plasmon esonance (SPR)-based senso was de eloped using epi ope-media ed HER2-nanoMIPs. The nanoMIP ecep o s we e cha ac e ized using dynamic ligh sca e ing (DLS), ze a po en ial, Fou ie - ans o m in a ed spec oscopy (FT-IR), ansmission elec on mic oscopy (TEM), ene gy- dispe si e X- ay spec oscopy (EDX), and luo escen mic oscopy. The a e age size o he nanoMIPs was de e mined o be 67.5 ±12.5 nm. The p oposed no el SPR senso p o ided supe io selec i i y o HER2 wi h a de ec ion limi (LOD) o 11.6 pg mL −1 in human se um. The high speci ici y o he senso was con i med by c oss- eac i i y s udies using P53, human se um albumin (HSA), ans e in, and glucose. The senso p epa a ion s eps we e success ully cha ac e ized by employing cyclic and squa e wa e ol amme y. The nanoMIP–SPR senso demons a es g ea po en ial o use in he ea ly diagnosis o b eas cance as a obus ool wi h high sensi i i y, selec i i y, and speci ici y. Keywo ds: SPR senso ; human epide mal g ow h ac o ecep o 2 (HER2); pep ide imp in ing; nanoMIPs; b eas cance diagnosis 1. In oduc ion B eas cance (BC) is conside ed one o he leading causes o dea h in emales, and he BC-caused mo ali y a e has been inc easing annually [ 1 , 2 ]. Despi e he high p e alence and mo ali y caused by BC, i s ea ly ecogni ion and diagnosis can signi ican ly inc ease he su i al a e. Hence, i is necessa y o de elop suscep ible inno a i e me hods o p e en i e and he apeu ic measu es o inc ease he su i al a e o pa ien s wi h BC [ 2 ]. Human epide mal g ow h ac o ecep o -2 (HER2) is a ansmemb ane y osine kinase ecep o and plays a i al ole in egula ing a e age cell g ow h, di e en ia ion, and su i al [ 3 , 4 ]. The o e exp ession o HER2 is associa ed wi h a molecula anomaly in 15–25% o pa ien s wi h BC. Acco dingly, HER2 is conside ed a p ognos ic and p edic i e bioma ke o he de ec ion and moni o ing o b eas cance [5]. The exp ession o HER2 has been ecen ly assessed on he basis o a se o in asi e echniques, such as immunohis ochemis y (IHC) and biopsy using luo escen in si u hyb idiza ion (FISH) [ 6 – 9 ]. The mos impo an disad an ages o hese me hods a e di i- cul ies due o hei complex na u e and mul i-s ep p ocedu es [ 10 ], high cos , he necessi y o he long- e m assessmen o high-quali y issue samples [ 11 , 12 ], and he equi emen o ained pe sonnel. Enzyme-linked immunoso ben assay (ELISA) is gene ally used o de ec HER2 in he se um. Howe e , ELISA has some d awbacks, such as supp essing he - modynamic/kine ic s udies o an ibody–an igen in e ac ion and using labeled molecules o Mic omachines 2023,14, 1086. h ps://doi.o g/10.3390/mi14051086 h ps://www.mdpi.com/jou nal/mic omachines Mic omachines 2023,14, 1086 2 o 16 induce alse-posi i e esponses [ 13 ]. The e o e, he e is s ill a s ong need o de elop a apid, easy- o-use, nonin asi e, inexpensi e, and ul a-sensi i e me hod o de ec ing he HER2 bioma ke wi h he pu pose o minimizing he echnical impedimen s o con en ional me hods. Such a me hod may also accu a ely iden i y HER2 in he bloods eam [1]. To da e, a ious elec ochemical, SPR, piezoelec ic, and FRET-based op ical biosenso s ha a e cheape and mo e sensi i e han se ological me hods ha e been epo ed o he de ec ion o HER2 [ 1 ]. These biosenso s enable he selec i e and as de ec ion o HER2 wi h a low de ec ion limi [ 14 ]. Howe e , he ma e ials used as bio- ecep o s, including ap ame s [ 14 ], an ibodies [ 15 ], and pep ides [ 16 ], possess some disad an ages, such as being uns able and expensi e and ha ing a sho shel -li e, leading o di icul ies in in eg a ing biosenso sys ems. Recen ly, molecula ly imp in ed polyme s (MIPs), possible al e na i es o na u al ecogni ion elemen s, ha e inc easingly been used in biosensing and ha e ou s anding ad an ages, such as bea ing highly speci ic, sensi i e, s abile bio ecogni ion ca i ies on he senso su aces; obus ness; esis ance o ex eme physical condi ions; and being syn hesized ia ela i ely simple, cheap, and scaleable p o ocols [ 17 , 18 ]. In he molecula imp in ing ield, epi ope imp in ing is gene ally p e e ed o e whole-p o ein imp in ing because o he p oblems a ising om he la ge, complex s uc u e o p o eins as well as he changes in hei con o ma ion du ing he imp in ing p ocess [ 19 , 20 ]. Epi ope-imp in ed MIPs a e able o ecognize bo h he epi ope and whole mac omolecules (e.g., p o eins, i uses, and bac e ia) and bind o hem speci ically [ 21 ]. The solid-phase syn hesis app oach o ob aining nanoMIPs esul s in he o ma ion o high-a ini y and -sensi i i y ecep o s e en in aqueous media, which is he na u al en i onmen o biological molecules. In addi ion o his, i allows low de ec ion limi s o de eloped senso s [ 22 ]. NanoMIPs a e p omising al e na i es o na u al an ibodies in diagnos ic and in i o applica ions due o hei cos e iciency, high a ini y, and s abili y [23–25]. To ou knowledge, in his s udy, a minia u ized SPR-based senso was de eloped o he i s ime o he de ec ion o HER2 using epi ope-media ed nanoMIPs. The nanoMIP ecep o s we e cha ac e ized using dynamic ligh sca e ing (DLS), ze a po en ial, Fou ie - ans o m in a ed spec oscopy (FT-IR), ansmission elec on mic oscopy (TEM), ene gy- dispe si e X- ay spec oscopy (EDX), luo escence mic oscopy analyses, and elec ochemi- cal me hods. The nanoMIP–SPR senso could de ec HER2 wi h high selec i i y, speci ici y, and sensi i i y. 2. Ma e ials and Me hods 2.1. Reagen s and Chemicals N-isop opylac ylamide (NIPAm), N,N 0 -me hylenebisac ylamide (BIS), N-(3-aminop opyl) me hac ylamide hyd ochlo ide (APMA), ac ylic acid (AAc), N- e -bu ylac ylamine (TBAm), N,N,N 0 ,N 0 - e ame hyle hylenediamine (TEMED), ammonium pe sulpha e (APS), e hanol (absolu e), me hanol (ACS eagen , ≥ 99.8%), ace one (ACS eagen , ≥ 99.5%), oluene (anhy- d ous), glu a aldehyde (GA), e hanolamine, Tween 20 (polyoxye hyleneso bi an monolau a e), phospha e-bu e ed saline (PBS), sul u ic acid, hyd ogen pe oxide, 11-me cap oundecanoic acid (MUDA), N-hyd oxysuccinimide (NHS), 1-e hyl-3-(3-dime hylaminop opyl) ca bodiimide (EDC), sodium hyd oxide (NaOH), sodium bo ohyd ide (SBH), ans e in om human blood plasma, albumin om human se um (HSA), and umo p o ein p53 we e pu chased om Sigma Ald ich Chemical Co. (Hambu g, Ge many). 3-Aminop opyl ie hoxysilane (APTES) was ob ained om Fishe Scien i ic (Schwe e, Ge many). Me hac yloxye hyl hioca bamoyl hodamine B was p o ided by Polysciences Eu ope (Be gs asse, Ge many). HER2-ECD (10004-HCCH) and he pep ide (163–175: DTNRSRACHPCSP) chosen o imp in ing we e p o ided by Sino Biological (Eschbo n, Ge many) and GenSc ip Bio ech (Nanjing, China), espec i ely. Glass beads (0.75–1.0 mm), glucose, and sy inge il e s (Ro i- labo PTFE, 0.45 µ m and 0.22 µ m) we e supplied by Ca l Ro h (Ka ls uhe, Ge many). Wi h- ou addi ional pu i ica ion, all compounds and sol en s we e o analy ical o HPLC quali y. A 0.22 µ m sy inge il e was used o il e a phospha e-bu e ed saline + 0.05% Tween Mic omachines 2023,14, 1086 3 o 16 (PBS/T) bu e . Double-dis illed wa e (DDW, p oduced by Ion Ex Mischbe pa one, A no Wille s, Hambu g, Ge many) was used o p epa e all solu ions in he aqua ic en i onmen . 2.2. P epa a ion o Glass Beads o NanoMIP Syn hesis Fi s , he glass beads, he i m suppo o he solid-phase syn hesis, we e p epa ed o he silaniza ion p ocess. Fo his, 60 g o glass beads was weighed in a beake and ac i a ed by boiling in 2 M NaOH solu ion o 15 min. Then, he glass beads we e washed i e imes wi h DDW, ou imes wi h PBS (pH: 7.4), and i e imes wi h DDW. A e he las wash wi h excess wa e , he pH alue o he wash wa e was de e mined o be 7.0–7.5, and he p ocess was con inued by washing he glass beads wice wi h ace one. The beads we e hen d ied in a ni ogen a mosphe e and incuba ed o e nigh in 2% / APTES solu ion (in anhyd ous oluene) in a sealed con aine . A e his s ep, he silaniza ion pa o he p ocess was comple ed. The glass beads we e emo ed om he APTES solu ion and washed ou imes wi h ace one and me hanol. Beads d ied wi h ni ogen gas we e incuba ed in a solu ion o 7% GA p epa ed in PBS (pH: 7.4) o 120 min. This is an in e media e s ep so he pep ide can be co alen ly a ached o he glass beads. Following his s ep, 10 mg o he cold pep ide solu ion dissol ed in 40 mL PBS was added o he glass beads, which we e washed i e imes wi h DDW. This is an essen ial s ep in he p ocess, and o e nigh incuba ion was allowed o ensu e ha he pep ide was co alen ly bound o he beads. A e incuba ion, he pep ide solu ion was pou ed ou , and he glass beads we e washed i e imes wi h DDW. A e wa d, he glass beads we e ea ed wi h SBH solu ion (1 mg mL −1 ) p epa ed in PBS (pH: 7.4) o 30 min. SBH is an e ec i e aldehyde-blocking agen wi h a s ong educing s uc u e [ 22 ]. Glass beads, epea edly washed wi h an excess o DDW, in e ac ed wi h 50 mL o 0.1 mM e hanolamine (pH: 7.4 in PBS) solu ion o 15 min o a oid non-speci ic in e ac ions and sel - eac ion o unconjuga ed ee GA g oups. A e he o med in e ac ion, he e hanolamine solu ion was pou ed ou , and he glass beads we e washed i e imes wi h DDW, d ied in a ni ogen a mosphe e, and placed in he eac ion lask. A e his s ep, he glass beads we e eady o nanoMIP syn hesis (Figu e 1). 2.3. Syn hesis o Ta ge NanoMIPs The necessa y monome s o polyme iza ion we e i s mixed. In b ie , 39 mg o NI- PAm, 2 mg o BIS, 54 mg o APMA, and 2.2 µ L o AAc we e added o 98 mL o PBS ( pH: 7.4 ) solu ion in a eac ion lask. In addi ion, 38 mg o TBAm and 3 mg o me hac yloxye hyl hioca bamoyl hodamine B monome s we e sepa a ely dissol ed in 1 mL o absolu e e hanol and added o he monome mix u e in he eac ion lask. The monome mix u e was s i ed unde a magne ic s i e o 30 min. A e wa d, he mix u e was sonica ed (T anssonic Digi al S, Elma Schmidbaue GmbH, Singen, Ge many) o 20 min, and hen ni ogen gas was passed h ough he mix u e o 20 min. The p ocess was con inued by adding pep ide-coupled glass beads o he eac ion lask, and ni ogen gas was e-passed ia he mix u e o 2 min. Then, 800 µ L o APS solu ion (ini ia o , 60 mg mL −1 , in wa e ) and 24 µ L o TEMED (ac i a o ) we e ins an ly added o he monome mix u e, ni ogen gas was passed h ough he mou h o he eac ion lask o 30 s, and he lask was igh ly closed wi h a sc ew cap. Polyme iza ion was comple ed in 1 h, he bo le was opened, and he cold wash and ho wash s ages we e ca ied ou one a e he o he . Ho and cold wash- ing s eps we e applied in a polyp opylene SPE ube (including polye hylene i s: 20 µm po e size). The cold wash p ocess was pe o med o emo e un eac ed monome s and low-a ini y nanoMIPs om he polyme iza ion medium. Fo his s ep, h ee consecu i e washes we e ca ied ou wi h 20 mL o DDW (5 ◦ C). Then, he SPE column con aining he glass beads was kep in a wa e ba h (GFL Shaking Wa e Ba h 1083, Bu gwedel, Ge many) o 15 min o p epa e o ho washing. In he nex s ep, se en washes we e pe o med wi h 20 mL o DDW (65 ◦ C) each o collec he high-a ini y ( a ge ) nanoMIPs (Figu e 2). The collec ed nanoMIP pool (140 mL) was s o ed a 4 ◦ C o he subsequen expe imen al s ud- ies. Th ee samples o 10 mL we e aken o calcula e he yield om he ob ained nanoMIP suspension. These samples we e placed in he p ede e mined weigh glass ials and d ied Mic omachines 2023,14, 1086 4 o 16 using eeze-d ying (ALPHA 2–4LD Plus eeze-d ye , Ch is , Os e ode am Ha z, Ge many) o 48 h. A e comple ely emo ing he wa e h ough he d ying p ocess, he glass ials we e e-weighed, and he weigh o he nanoMIPs was de e mined by sub ac ing he a e o he ials om he ob ained weigh alues. Mic omachines 2023, 14, x FOR PEER REVIEW 4 o 17 Figu e 1. The immobiliza ion o he HER2-pep ide on glass beads as he empla e. 2.3. Syn hesis o Ta ge NanoMIPs The necessa y monome s o polyme iza ion we e i s mixed. In b ie , 39 mg o NIPAm, 2 mg o BIS, 54 mg o APMA, and 2.2 µL o AAc we e added o 98 mL o PBS (pH: 7.4) solu ion in a eac ion lask. In addi ion, 38 mg o TBAm and 3 mg o me hac yloxye hyl hioca bamoyl hodamine B monome s we e sepa a ely dissol ed in 1 mL o absolu e e hanol and added o he monome mix u e in he eac ion lask. The monome mix u e was s i ed unde a magne ic s i e o 30 min. A e wa d, he mix u e was sonica ed (T anssonic Digi al S, Elma Schmidbaue GmbH, Singen, Ge many) o 20 min, and hen ni ogen gas was passed h ough he mix u e o 20 min. The p ocess was con inued by adding pep ide-coupled glass beads o he eac ion lask, and ni ogen gas was e-passed ia he mix u e o 2 min. Then, 800 µL o APS solu ion (ini ia o , 60 mg mL −1 , in wa e ) and 24 µL o TEMED (ac i a o ) we e ins an ly added o he monome mix u e, ni ogen gas was passed h ough he mou h o he eac ion lask o 30 s, and he lask was igh ly closed wi h a sc ew cap. Polyme iza ion was comple ed in 1 h, he bo le was opened, and he cold wash and ho wash s ages we e ca ied ou one a e he o he . Ho and cold washing s eps we e applied in a polyp opylene SPE ube (including polye hylene i s: 20 µm po e size). The cold wash p ocess was pe o med o emo e un eac ed monome s and low-a ini y nanoMIPs om he polyme iza ion medium. Fo his s ep, h ee consecu i e washes we e ca ied ou wi h 20 mL o DDW (5 °C). Then, he Figu e 1. The immobiliza ion o he HER2-pep ide on glass beads as he empla e. 2.4. Cha ac e iza ion o NanoMIPs The size dis ibu ion p o iles o nanoMIPs we e de e mined by a dynamic ligh sca e - ing (DLS) de ice (Mal e n Panaly ical, Ze asize P o., He enbe g, Ge many). Six y uns we e pe o med in he backsca e ed mode o each eco ding. In addi ion, he ze a po en ial alue was de e mined wi h he same de ice o measu e he s abili y o he nanoMIPs in wa- e . The sample o he nanoMIP solu ion, which was d opped on he glass slide and d ied, was imaged wi h a luo escence mic oscope (BZ-X800LE, Keyence, Neu-Isenbu g, Ge - many). The mic oscope (BZ-PA10, Plan Apoch oma 10X, NA 0.45, WD 4 mm) had a 40 W LED luo escen ligh sou ce and a powe supply o 100 o 240 VAC ± 10%, 50/60 Hz . O no e, he luo escen p ope y o me hac yloxye hyl hioca bamoyl hodamine B monome (exci a ion max: 548 nm, concen a ion in he polyme iza ion mix u e: 45 µ M) ga e he polyme a luo escen na u e. An FT-IR de ice (Ca y 630 FTIR, Agilen Technologies, San a Cla a, CA, USA) was used o analyze he unc ional g oups ha exis ed in nanoMIPs. TEM analyses we e pe o med on an FEI Tecnai F30 G2 STwin (300 kV, FEG) equipped wi h an Mic omachines 2023,14, 1086 5 o 16 EDX de ec o (Si/Li, EDAX) o isualize he mo phological s uc u e o he nanoMIPs and analyze he elemen al composi ion o he polyme . Mic omachines 2023, 14, x FOR PEER REVIEW 5 o 17 SPE column con aining he glass beads was kep in a wa e ba h (GFL Shaking Wa e Ba h 1083, Bu gwedel, Ge many) o 15 min o p epa e o ho washing. In he nex s ep, se en washes we e pe o med wi h 20 mL o DDW (65 °C) each o collec he high-a ini y ( a ge ) nanoMIPs (Figu e 2). The collec ed nanoMIP pool (140 mL) was s o ed a 4 °C o he subsequen expe imen al s udies. Th ee samples o 10 mL we e aken o calcula e he yield om he ob ained nanoMIP suspension. These samples we e placed in he p ede e mined weigh glass ials and d ied using eeze-d ying (ALPHA 2–4LD Plus eeze-d ye , Ch is , Os e ode am Ha z, Ge many) o 48 h. A e comple ely emo ing he wa e h ough he d ying p ocess, he glass ials we e e-weighed, and he weigh o he nanoMIPs was de e mined by sub ac ing he a e o he ials om he ob ained weigh alues. Figu e 2. The syn hesis p inciple o pep ide-imp in ed nanoMIPs. 2.4. Cha ac e iza ion o NanoMIPs The size dis ibu ion p o iles o nanoMIPs we e de e mined by a dynamic ligh sca e ing (DLS) de ice (Mal e n Panaly ical, Ze asize P o., He enbe g, Ge many). Six y uns we e pe o med in he backsca e ed mode o each eco ding. In addi ion, he ze a po en ial alue was de e mined wi h he same de ice o measu e he s abili y o he nanoMIPs in wa e . The sample o he nanoMIP solu ion, which was d opped on he glass slide and d ied, was imaged wi h a luo escence mic oscope (BZ-X800LE, Keyence, Neu- Isenbu g, Ge many). The mic oscope (BZ-PA10, Plan Apoch oma 10X, NA 0.45, WD 4 Figu e 2. The syn hesis p inciple o pep ide-imp in ed nanoMIPs. In addi ion, he success ul de elopmen o he nanoMIP senso was e i ied using wo main elec ochemical echniques, including cyclic ol amme y (CV) and squa e-wa e ol amme y (SWV). Fo his, a gold subs a e was used as he wo king elec ode in he elec ochemical measu emen se up (PalmSens4 wo ks a ion, Bell ec, Lüdenscheid, Ge many). All CV measu emen s we e pe o med a a po en ial ange o − 0.2 o 0.8 V and a scan a e o 0.05 V s −1 . The ange o applied po en ials o SWV measu emen s was − 0.3 o 0.8 V a an ampli ude o 0.05 V and a equency o 5 o 10 Hz. The expe imen s we e pe o med a oom empe a u e. 2.5. Op ical De ec ion o he HER2 Bioma ke The ba e gold SPR chip was ini ially cleaned wi h a mix u e o hyd ogen pe oxide (35%, 2 mL), ammonia (25%, 2 mL), and millipo e wa e (50 mL) and boiled a 80 ◦ C o 20 min . Fo his p ocess, wo gold chips we e placed in a chip holde and imme sed in he p ehea ed mix u e. Mos con aminan s on he gold su ace we e elimina ed using pi anha solu ion (a 3:1 mix u e o sul u ic acid and 30% hyd ogen pe oxide). A e his ea men , he gold chips we e washed i e imes wi h millipo e wa e and h ee imes wi h absolu e e hanol. The chips we e hen d ied wi h a gen le low o ni ogen gas. Nex , he gold chips we e imme sed in he MUDA solu ion (2 mM, 5 mL) p epa ed in absolu e e hanol in a Pe i dish and incuba ed o e nigh in he da k. This p ocess c ea ed a sel -assembled monolaye on he su ace o he chips. A e incuba ion, he gold chips we e washed wi h an excess o Mic omachines 2023,14, 1086 6 o 16 absolu e e hanol and double-dis illed wa e , gen ly d ied wi h ni ogen gas, and s o ed in a idge a 4 ◦C un il he ime o use. A minia u ized angula SPR de ice (CORGI IIF, Plasme ix, Mon eal, QC, Canada) was used o all de ec ion s udies, including HER-2 pep ide and p o ein as he a ge molecules. Du ing he senso expe imen s, he solu ions we e allowed o pass h ough he chip su ace wi h a pe is al ic pump (Isma ec Reglo ICC Digi al pump, 2-channel, Cole-Pa me GmbH, We heim, Ge many), p o iding a low a e o 4 µ L min −1 . MUDA- coa ed chips we e ac i a ed wi h a 4 min injec ion o a eshly made EDC/NHS solu ion ( 0.4 M EDC , 0.1 M NHS) o he co alen immobiliza ion o he nanoMIPs wi h he aid o amine coupling chemis y. A cons an low a e (4 µ L min −1 ) was main ained o 8 min o immobilize he nanoMIPs on he gold chip su ace. To p epa e he nanoMIP medium ( 500 µg mL−1 ), he suspension was p epa ed wi h degassed PBS/T and il e ed h ough a 0.22 µm sy inge il e p io o sonica ion o 30 min. The samples we e p epa ed in PBS/T o p omo e luid low h ough he senso and mic o ube channels and p e en ai om becoming apped inside he mic o luidics. The nanoMIP suspension was hen e- il e ed wi h a 0.45 µ m sy inge il e . The chip su ace hen in e ac ed wi h 1.0 mM e hanolamine solu ion o 4 min o block possible ac i e si es ha may ha e emained on he su ace a e nanoMIP immobiliza ion. The injec ion o samples, including HER2 pep ide and HER2 bioma ke , a a ying concen a ions we e subsequen ly ca ied ou (Figu e 3). The associa ion and dissocia ion imes we e se a 5 and 2 min, espec i ely. Mic omachines 2023, 14, x FOR PEER REVIEW 7 o 17 Figu e 3. The p epa a ion o nanoMIP-based senso sys em. 3. Resul s and Discussion 3.1. Size and S abili y o Ta ge NanoMIPs The hyd odynamic size o a ge nanoMIPs was measu ed in DDW a oom empe a u e by employing DLS. The nanoMIPs we e disco e ed o ha e an a e age hyd odynamic adius o 97.79 ± 0.53 nm and a polydispe si y index (PDI) o 0.263, e ealing ema kably uni o m and monodispe se pa icles (Figu e 4a,b). Addi ionally, he ze a po en ial o he nanoMIPs in DDW was es ed o asce ain he s abili y o he solu ion. The a e age ze a po en ial o nanopa icles (NPs) was ound o be −12.37 ± 0.32 mV (Figu e 4c). I was hypo hesized ha he NPs o ming a dispe sed phase endowed he solu ion wi h a colloidal cha ac e . Figu e 3. The p epa a ion o nanoMIP-based senso sys em. Mic omachines 2023,14, 1086 7 o 16 Fo selec i i y s udies, a con ol nanoMIP was ob ained by imp in ing a di e en pep ide (ISASRKLQLK). I was immobilized on he senso su ace by ollowing he a o e- men ioned p ocedu e p io o he injec ion o a ge analy es o he de e mina ion o senso selec i i y. Fu he mo e, he speci ici y o he de eloped nanoMIP–SPR senso was ealized by s udying e e ence molecules (i.e. P53, HSA, ans e in, and glucose). 3. Resul s and Discussion 3.1. Size and S abili y o Ta ge NanoMIPs The hyd odynamic size o a ge nanoMIPs was measu ed in DDW a oom empe a- u e by employing DLS. The nanoMIPs we e disco e ed o ha e an a e age hyd odynamic adius o 97.79 ± 0.53 nm and a polydispe si y index (PDI) o 0.263, e ealing ema k- ably uni o m and monodispe se pa icles (Figu e 4a,b). Addi ionally, he ze a po en ial o he nanoMIPs in DDW was es ed o asce ain he s abili y o he solu ion. The a e age ze a po en ial o nanopa icles (NPs) was ound o be − 12.37 ± 0.32 mV (Figu e 4c). I was hypo hesized ha he NPs o ming a dispe sed phase endowed he solu ion wi h a colloidal cha ac e . Mic omachines 2023, 14, x FOR PEER REVIEW 8 o 17 (a) (b) (c) Figu e 4. (a) The hyd odynamic size dis ibu ion o a ge nanoMIPs; (b) The co ela ion i o DLS analysis; (c) The ze a po en ial p o ile o nano-polyme s. 3.2. Fluo escence Mic oscopy and TEM Analyses Fo luo escence mic oscopy imaging, a ce ain concen a ion (500 µg mL −1 ) o nanoMIP solu ions was p epa ed. The luo escence mic oscopy image p o ed ha he luo escen monome (me hac yloxye hyl hioca bamoyl hodamine B) used in he syn hesis o nanoMIP was well inco po a ed in o he polyme ic s uc u e (Figu e 5a). Addi ionally, TEM images con i med he exac size, shape, and uni o mi y o he nanoMIPs. (Figu e 5b). The size o pa icles acqui ed by TEM was app oxima ely 67.5 ± 12.5 nm which was smalle han hose measu ed by DLS due o sol a ion and swelling o polyme pa icles in he la e case. Ano he eason is ha he agglome a ion o nanoMIPs in solu ion causes an e iden size inc emen in DLS measu emen s [26]. Figu e 4. ( a ) The hyd odynamic size dis ibu ion o a ge nanoMIPs; ( b ) The co ela ion i o DLS analysis; (c) The ze a po en ial p o ile o nano-polyme s. Mic omachines 2023,14, 1086 8 o 16 3.2. Fluo escence Mic oscopy and TEM Analyses Fo luo escence mic oscopy imaging, a ce ain concen a ion (500 µ g mL −1 ) o nanoMIP solu ions was p epa ed. The luo escence mic oscopy image p o ed ha he luo- escen monome (me hac yloxye hyl hioca bamoyl hodamine B) used in he syn hesis o nanoMIP was well inco po a ed in o he polyme ic s uc u e (Figu e 5a). Addi ionally, TEM images con i med he exac size, shape, and uni o mi y o he nanoMIPs. (Figu e 5b). The size o pa icles acqui ed by TEM was app oxima ely 67.5 ± 12.5 nm which was smalle han hose measu ed by DLS due o sol a ion and swelling o polyme pa icles in he la e case. Ano he eason is ha he agglome a ion o nanoMIPs in solu ion causes an e iden size inc emen in DLS measu emen s [26]. Mic omachines 2023, 14, x FOR PEER REVIEW 9 o 17 (a) (b) Figu e 5. (a) Fluo escence mic oscopy and (b) TEM images o nanoMIPs. 3.3. FT-IR and EDX Analysis FT-IR analysis was pe o med o p o e he success ul syn hesis o pep ide-imp in ed nanoMIPs (Figu e 6a,b). The esul s e ealed ha he spec um o NIPAM as a s uc u al monome was signi ican ly di e en om he polyme (nanoMIP) spec um and exhibi ed only a ew expec ed peaks. C-H asymme ic s e ching (2967 cm−1), C-H symme ic s e ch- ing (2877 cm−1), C=O amide g oup (1632 cm−1), N-H bending (1545 cm−1), C-N s e ching (1366 cm−1), -CH2 bending (1452 cm−1), and -CH3 bending (1385 cm−1) ib a ions we e no able peaks in he spec um o NIPAM. Vib a ions such as C-H asymme ic–symme ic s e ching (2917 cm−1 and 2848 cm−1, espec i ely), C=O amide g oup (1636 cm−1), and N- H bending (1539 cm−1) also appea ed in he polyme spec um. In addi ion, he FT-IR analysis o he imp in ed pep ide e ealed ha no peak o he pep ide was iden i ied in he nanoMIP spec um. This indica es ha he empla e emo al om he polyme ic s uc u e, as one o he mos c i ical s eps o he imp in ing p ocess, was e ec i ely achie ed. Fu he mo e, he EDX analysis was pe o med o analyze he elemen al composi ion o nanoMIPs. Ca bon and oxygen elemen s in he s uc u e indica ed he syn hesis o he desi ed o ganic polyme . (Figu e 6c). Figu e 5. (a) Fluo escence mic oscopy and (b) TEM images o nanoMIPs. 3.3. FT-IR and EDX Analysis FT-IR analysis was pe o med o p o e he success ul syn hesis o pep ide-imp in ed nanoMIPs (Figu e 6a,b). The esul s e ealed ha he spec um o NIPAM as a s uc u al monome was signi ican ly di e en om he polyme (nanoMIP) spec um and exhibi ed only a ew expec ed peaks. C-H asymme ic s e ching (2967 cm −1 ), C-H symme ic s e ch- ing (2877 cm −1 ), C=O amide g oup (1632 cm −1 ), N-H bending (1545 cm −1 ), C-N s e ching (1366 cm −1 ), -CH 2 bending (1452 cm −1 ), and -CH 3 bending (1385 cm −1 ) ib a ions we e no able peaks in he spec um o NIPAM. Vib a ions such as C-H asymme ic–symme ic s e ching (2917 cm −1 and 2848 cm −1 , espec i ely), C=O amide g oup (1636 cm −1 ), and Mic omachines 2023,14, 1086 9 o 16 N-H bending (1539 cm −1 ) also appea ed in he polyme spec um. In addi ion, he FT-IR analysis o he imp in ed pep ide e ealed ha no peak o he pep ide was iden i ied in he nanoMIP spec um. This indica es ha he empla e emo al om he polyme ic s uc u e, as one o he mos c i ical s eps o he imp in ing p ocess, was e ec i ely achie ed. Mic omachines 2023, 14, x FOR PEER REVIEW 10 o 17 (a) (b) (c) Figu e 6. The compa ison o FT-IR spec um o nanoMIPs wi h FT-IR spec um o (a) NIPAM; (b) imp in ed pep ide; (c) he elemen al composi ion o nanoMIPs. Figu e 6. The compa ison o FT-IR spec um o nanoMIPs wi h FT-IR spec um o ( a ) NIPAM; (b) imp in ed pep ide; (c) he elemen al composi ion o nanoMIPs. Mic omachines 2023,14, 1086 16 o 16 10. Goh ing, J.T.; Dale, P.S.; Fan, X. De ec ion o HER2 b eas cance bioma ke using he op o- luidic ing esona o biosenso . Sens. Ac ua o s B Chem. 2010,146, 226–230. [C ossRe ] 11. Hanash, S.; Taguchi, A. Applica ion o p o eomics o cance ea ly de ec ion. Cance J. 2011,17, 423–428. [C ossRe ] 12. Shukla, S.; Singh, B.K.; Pa hania, O.P.; Jain, M. E alua ion o HER2/neu oncop o ein in se um & issue samples o women wi h b eas cance . Ind. J. Med. Res. 2016,143 (Suppl. S1), S52–S58. 13. Mon ei o, J.P.; P edabon, S.M.; Bona é, E.G.; Ma ins, A.F.; B olo, A.G.; Rado ano ic, E.; Gi o o, E.M. SPR pla o m based on image acquisi ion o HER2 an igen de ec ion. Nano echnology 2016,28, 045206. [C ossRe ] [PubMed] 14. Zhang, Y.; Xu, Y.; Li, N.; Qi, N.; Peng, L.; Yang, M.; Hou, C.; Huo, D. An ul asensi i e dual-signal a io elec ochemical ap ame biosenso o he de ec ion o HER2. Colloids Su . B 2023,222, 113118. [C ossRe ] [PubMed] 15. Ele xige a, U.; Ma inez-Pe digue o, J.; Ba de as, R.; Pinga ón, J.M.; Campuzano, S.; Me ino, S. Su ace plasmon esonance immunosenso o E bB2 b eas cance bioma ke de e mina ion in human se um and aw cance cell lysa es. Anal. Chim. Ac a 2016,905, 156–162. [C ossRe ] 16. Lu, L.; Liu, X.; Zuo, C.; Zhou, J.; Zhu, C.; Zhang, Z.; Fille , M.; C ommen, J.; Jiang, Z.; Wang, Q. In i o / in i o deg ada ion analysis o as uzumab by combining speci ic cap u e on HER2 mimo ope pep ide modi ied ma e ial and LC-QTOF-MS. Anal. Chim. Ac a 2022,1225, 340199. [C ossRe ] 17. Hassine, A.B.; Raoua i, N.; Mo ei a, F.T. No el biomime ic P ussian blue nanocubes-based biosenso o Tau-441 p o ein de ec ion. J. Pha m. Biomed. Anal. 2023,226, 115251. [C ossRe ] 18. Bajaj, A.; T impe , J.; Abdulhalim, I.; Al in as, Z. Syn hesis o molecula ly imp in ed polyme nanopa icles o SARS-CoV-2 i us de ec ion using su ace plasmon esonance. Chemosenso s 2022,10, 459. [C ossRe ] 19. Al in as, Z.; Takiden, A.; U esch, T.; M oginski, M.A.; Schmid, B.; Schelle , F.W.; Süssmu h, R.D. In eg a ed App oaches owa d High-A ini y A i icial P o ein Binde s Ob ained ia Compu a ionally Simula ed Epi opes o P o ein Recogni ion. Ad . Func . Ma e . 2019,29, 1807332. [C ossRe ] 20. Tchinda, R.; Tu sch, A.; Schmid, B.; Süssmu h, R.D.; Al in as, Z. Recogni ion o p o ein bioma ke s using epi ope-media ed molecula ly imp in ed ilms: His idine o cys eine modi ied epi opes? Biosens. Bioelec on. 2019,123, 260–268. [C ossRe ] 21. Ansa i, S.; Masoum, S. Molecula ly imp in ed polyme s o cap u ing and sensing p o eins: Cu en p og ess and u u e implica ions. T ends Analy . Chem. 2019,114, 29–47. [C ossRe ] 22. Choudha y, S.; Al in as, Z. De elopmen o a Poin -o -Ca e SPR Senso o he Diagnosis o Acu e Myoca dial In a c ion. Biosenso s 2023,13, 229. [C ossRe ] [PubMed] 23. Yang, J.C.; Cho, C.H.; Choi, D.Y.; Pa k, J.P.; Pa k, J. Mic ocon ac su ace imp in ing o a ini y pep ide o elec ochemical impedime ic de ec ion o neu ophil gela inase-associa ed lipocalin. Sens. Ac ua o s B Chem. 2022,364, 131916. [C ossRe ] 24. Case a, G.; Zhang, X.; Ya man, A.; Supala, E.; Wollenbe ge , U.; Gyu csányi, R.E.; Zebge , I.; Schelle , F.W. Insigh s in elec osyn- hesis, a ge binding, and s abili y o pep ide-imp in ed polyme nano ilms. Elec ochim. Ac a 2021,381, 138236. [C ossRe ] 25. Tan, F.; Zhai, M.; Meng, X.; Wang, Y.; Zhao, H.; Wang, X. Hyb id pep ide-molecula ly imp in ed polyme in e ace o elec o- chemical de ec ion o ancomycin in complex ma ices. Biosens. Bioelec on. 2021,184, 113220. [C ossRe ] 26. Can a o a, F.; Czulak, J.; Be lem, K.; Sachde a, A.; Ee sels, K.; Van G ins en, B.; Cleij, T.; Pee e s, M. A no el he mal de ec ion me hod based on molecula ly imp in ed nanopa icles as ecogni ion elemen s. Nanoscale 2018,10, 2081–2089. [C ossRe ] 27. Al in as, Z. Su ace plasmon esonance based senso o he de ec ion o glycopep ide an ibio ics in milk using a ionally designed nanoMIPs. Sci. Rep. 2018,8, 11222. [C ossRe ] 28. Sypabeko a, M.; Aman aye a, A.; Vangelis a, L.; González-Vila, Á.; Cauche eu , C.; Tosi, D. Ul alow limi de ec ion o soluble HER2 bioma ke in se um wi h a ibe -op ic ball- ip esona o assis ed by a il ed FBG. ACS Meas. Sci. Au 2022 , 2, 309–316. [C ossRe ] 29. Sha ma, S.; Zapa e o-Rod íguez, J.; Saxena, R.; O’Kennedy, R.; S i as a a, S. Ul asensi i e di ec impedime ic immunosenso o de ec ion o se um HER2. Biosens. Bioelec on. 2018,106, 78–85. [C ossRe ] 30. Ha a i, Y.W.; Nu djanah, D.; Wyan u i, S.; Angg aeni, A.; Ga a , S. Gold nanopa icles modi ied sc een-p in ed immunosenso o cance bioma ke HER2 de e mina ion based on an i HER2 bioconjuga es. In P oceedings o he he 3 d In e na ional Semina On Chemis y: G een Chemis y and i s Role o Sus ainabili y, Su abaya, Indonesia, 18–19 July 2018; AIP Publishing LLC: Long Island, NY, USA, 2018. 31. Zhang, Y.; Li, N.; Xu, Y.; Liu, X.; Ma, Y.; Huang, Z.; Luo, H.; Hou, C.; Huo, D. A no el elec ochemical biosenso based on AMNFs@ ZIF-67 nano composi e ma e ial o ul asensi i e de ec ion o HER2. Bioelec ochemis y 2023 ,150, 108362. [C ossRe ] Disclaime /Publishe ’s No e: The s a emen s, opinions and da a con ained in all publica ions a e solely hose o he indi idual au ho (s) and con ibu o (s) and no o MDPI and/o he edi o (s). MDPI and/o he edi o (s) disclaim esponsibili y o any inju y o people o p ope y esul ing om any ideas, me hods, ins uc ions o p oduc s e e ed o in he con en .