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
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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 .