CYTOCHROME C PURIFICATION WITH SURFACE 
IMPRINTED BACTERIAL CELLULOSE NANOFIBERS 

 
 
 

YÜZEY BASKILANMIŞ BAKTERİYEL SELÜLOZ 
NANOFİBERLER İLE SİTOKROM C SAFLAŞTIRILMASI  

 
 
 

EMEL TAMAHKAR IRMAK 
 

 

 

PROF. DR. ADİL DENİZLİ 
Supervisor 

 

 

 

 

Submitted to Graduate School of Science and Engineering of Hacettepe University  

as a Partial Fulfillment to the Requirements  

for the Award of the Degree of Doctor of Philosophy 

In Bioengineering 

 

 

2014 

 



ETHICS 
 
In this thesis study, prepared in accordance with the spelling rules of Graduate 

School of Science and Engineering of Hacettepe University, 

 

I declare that 

 

• all	
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• all	
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• I	
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• and	
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this	
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19/12/2014 

 

 

EMEL TAMAHKAR IRMAK 

 
 
 
 
 
 
 
 
 
 



 i 

ÖZET 

 
 

YÜZEY BASKILANMIŞ BAKTERİYEL SELÜLOZ NANOFİBERLER 
İLE SİTOKROM C SAFLAŞTIRILMASI 

 
 

Emel TAMAHKAR IRMAK 
Doktora, Biyomühendislik Anabilim Dalı 
Tez Danışmanı: Prof. Dr. Adil DENİZLİ 

 İkinci Tez Danışmanı: Prof. Dr. Tülin KUTSAL 
Aralık 2014, 93 sayfa 

 
 

Bu çalışmada, sitkrom c saflaştırma ve tanıma işlemleri için yüzey baskılama 

yöntemi ile sitokrom c baskılanmış bakteriyel selüloz (Cyt c-MIP) nanofiberler 

hazırlanmıştır. N-metakriloil L-histidin metil ester (MAH) monomeri spesifik tanıma 

bölgeleri oluşturmak için sentezlenmiştir. MAH monomeri ile bakır (II) iyonu 

arasında kompleks oluşumundan sonra metal iyon koordinasyon etkileşimleri 

kullanılarak MAH-Cu(II) monomeri ve sitokrom c hedef molekülleri ile önkompleks 

hazırlanmıştır. Cyt c-MIP nanofiberler farklı % toplam monomer oranlarında, farklı 

monomer/hedef molekül molar oranlarında ve farklı polimerizasyon sürelerinde 

hazırlanmıştır. Cyt c-MIP nanofiberler ATR-FTIR, SEM ve temas açısı ölçümleri ile 

karakterize edilmiştir. Adsorplanan protein moleküllerinin polimer yapıdan 

desorpsiyonu için 1 M NaCl çözeltisi kullanılmıştır. Adsorpsiyon çalışmaları pH, 

sıcaklık ve iyonik şiddete göre gerçekleştirilmiştir ve adsorpsiyon kapasitesi 

spektrofotometrik olarak analiz edilmiştir. Uygun denge izotermi  Langmuir izotermi 

olarak belirlenmiştir. Bu sistem için hız sınırlayıcı basamağın kimyasal reaksiyon 

olmasından dolayı yalancı-ikinci derece kinetik modelin bu sisteme daha uygun 

olduğu belirlenmiştir. Cyt c-MIP nanofiberlerin seçiciliğinin değerlendirilmesi için 

hedef moleküle benzer yapıda olan sığır serum albumin, hemoglobin, miyoglobin 



 ii 

ve lizozim kullanılmıştır. Metal iyon koordinasyonu etkileşimlerinin spesifik 

bağlanma bölgeleri hazırlanmasında katkısının olduğu tespit edilmiştir. Bu 

sonuçlar hedef proteini baskılama işleminin yüksek adsorpsiyon kapasitesi ile 

başarılı bir şekilde gerçekleştiğini göstermiştir. Cyt c-MIP nanofiberlerin bağlanma 

özellikleri ayrıca QCM sensör çalışmaları ile de değerlendirilmiştir. Bu kapsamda 

QCM nanosensörler Cyt c-MIP nanofiberler ile hazırlanmış ve kinetic ve seçicilik 

çalışmalarında kullanılmıştır. Bu nanosensörler AFM ile karakterize edilmiştir. 

Bağlanma özelliklerinin belirlenmesi için kinetik ve bağlanma sabiti Scathard, 

Langmuir, Freundlich ve Langmuir-Freundlich izotermleri ile hesaplanmıştır. Elde 

edilen sonuçlar kesikli sistemde bağlanma çalışmalarının sonuçları ile 

örtüşmektedir.  

 

 

Anahtar kelimeler: Moleküler baskılanmış polimerler, yüzey baskılama, metal-

iyon koordinasyonu, protein tanıma, bakteriyel selüloz nanofiberler. 

 
 
 

 

 

 

 

 

 

 

 

 

 

 

 

 
 



 iii 

ABSTRACT 

 
 

CYTOCHROME C PURIFICATION WITH SURFACE IMPRINTED 
BACTERIAL CELLULOSE NANOFIBERS 

 
 

Emel TAMAHKAR IRMAK 
Doctor of Philosophy, Bioengineering Division 

Supervisor: Prof. Dr. Adil DENİZLİ 
Co-supervisor: Prof. Dr. Tülin KUTSAL 

December 2014, 93 pages 
 
 
 
In the present study, cytochrome c imprinted bacterial cellulose (Cyt c-MIP) 

nanofibers were prepared for the purification and recognition of cytochrome c via 

surface imprinting approach. N-methacryloyl-L-histidine methyl ester (MAH) was 

synthesized to create specific binding sites. After the complexation between MAH 

and chelating metal ion copper(II), the preorganized complex was prepared with 

MAH-Cu(II) monomer and Cyt c template molecules via metal ion coordination 

interactions. Cyt c-MIP nanofibers were prepared in the presence of different 

amounts of %w total monomer, monomer/template ratio and polymerization time. 

Cyt c-MIP nanofibers were characterized by ATR-FTIR, SEM and contact angle 

measurements. In order to desorp the adsorbed proteins from the polymer 

network, 1 M NaCl solution was used. Adsorption studies were performed with 

respect to pH, temperature and ionic strength and the adsorption capacity was 

evaluated spectrophotometrically. The suitable equilibrium isotherm was 

determined as Langmuir isotherm. It was determined that pseudo-second order 

kinetic model was more suitable to this system referring chemical reaction as a 

rate limiting step. To evaluate the selectivity of the Cyt c-MIP nanofibers, similar 

proteins were utilized as non-template proteins, which were bovine serum albumin, 



 iv 

hemoglobin, myoglobin and lysozyme. The metal ion coordination interactions 

were found to contribute for the fabrication of specific recognition binding sites. 

The results present the successful imprinting of the template protein with high 

adsorption capacity. The binding properties of Cyt c-MIP nanofibers were also 

evaluated with QCM sensor studies. In this context QCM nanosensors were 

prepared with Cyt c-MIP nanofibers and used for further kinetic and selectivity 

studies. These nanosensors were characterized by AFM. In order to determine the 

binding characteristics, kinetic and binding constant were calculated with 

Scathard, Langmuir, Freundlich and Langmuir-Freundlich isotherms. The results 

were conformable with the results of batch rebinding studies.  

 

 

Key words: Molecular imprinted polymers, surface imprinting, metal-ion 

coordination, protein recognition, bacterial cellulose nanofibers. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

 



 v 

ACKNOWLEDGEMENTS 

This thesis could not be finished without the help and support of many people who 

are gratefully acknowledged here. At the very first, I would like to express my 

deepest gratitude to my supervisor Prof. Dr. Adil Denizli for his patience, 

encouragement, support and supervision throughout my studies. I am gratefully 

indebted to him for many valuable discussions that helped me understand my 

research area better. 

I am also gratefully indebted to my co-supervisor Prof. Dr. Tülin Kutsal for 

encouragement, support and guidance. 

I would like to thank to my committee members, Prof. Dr. Serap Şenel, Prof. Dr. F. 

Sema Bektaş, Prof. Dr. Handan Yavuz Alagöz and Dr. Fatma Yılmaz for their 

contribution and comments for my thesis.  

A special thanks to Gülsu for the discussions, suggestions, her time and especially 

her friendship. In particular, I would like to thank to Müge and Işık for their help 

and support. Thanks to all my lab mates, Canan, Seda, Kemal, Fatma, Sevgi, 

Gizem, Mine, Çiğdem, Dilara, Yeşeren, Monir, Erdoğan, Emin, Aykut, Erkut, 

Recep, Esma, Nilay, Gözde, Ali, Bahar, Ilgım, Duygu, Semra, Sabina and Deniz, 

for their friendship, help and providing nice working environment.  

I would like to thank to Prof. Dr. Karsten Haupt for giving me an opportunity to 

work in his laboratory and for the warm hospitality during my stay in Compiegne. 

Thanks go to Serena Ambrosini for the contributions in my view of the academic 

life.  

I would like to express my warm thanks to my dear friends Bengi and Eda for their 

helpful advises, optimistic comments and particularly their friendship.  

My greatest thanks go to my family, to my dear mother Fatma and father Ertuğrul 

who always loved, encouraged and supported me.  

Finally, I would like to thank to my dear sister Ebru and my dear husband Sercan 

for their endless love, understanding and helping me get through the difficult times. 

To them I dedicated this thesis. 

 

 



 vi 

CONTENTS 

Page 
ÖZET ........................................................................................................................ i 
ABSTRACT ............................................................................................................. iii 
ACKNOWLEDGEMENTS ....................................................................................... v 
CONTENTS ............................................................................................................. vi 
FIGURES .............................................................................................................. viii 
TABLES ................................................................................................................... xi 
SYMBOLS AND ABBREVIATIONS ....................................................................... xii 
1. INTRODUCTION .............................................................................................. 1 
2. GENERAL INFORMATION .............................................................................. 4 
2.1. Molecular Imprinting Process ..................................................................... 4 
2.1. 1. Factors Affecting The Molecular Imprinting Process .................................... 5 
2.1. 2. Template ...................................................................................................... 5 
2.1. 3. Functional Monomer ..................................................................................... 5 
2.1. 4. Cross-linker .................................................................................................. 6 
2.1. 5. Initiation Method and Temperature .............................................................. 6 
2.1. 6. Polymerization Time ..................................................................................... 6 
2.1. 7. Solvent ......................................................................................................... 7 
2. 1. Types of Molecular Imprinting ......................................................................... 7 
2.2. 1. Covalent Imprinting ...................................................................................... 7 
2.2. 2. Non-covalent Imprinting ............................................................................... 8 
2.2. 3. Metal Ion Mediated Molecular Imprinting ..................................................... 8 
2. 2. Protein Imprinting .......................................................................................... 14 
2.3. 1. Bulk Imprinting ............................................................................................ 15 
2.3. 2. Surface Imprinting ...................................................................................... 16 
2. 3. Affinity Nanofiber Membranes for Protein Recognition ................................. 17 
2. 4. Bacterial Cellulose ......................................................................................... 25 
2. 5. Cytochrome c ................................................................................................ 27 
2. 6. QCM Sensors ................................................................................................ 29 
3. EXPERIMENTAL METHOD ........................................................................... 32 
3. 1. Materials ........................................................................................................ 32 
3. 2. Preparation of Cyt c-MIP Nanofibers ............................................................. 32 
3.2. 1.  Production of Bacterial Cellulose Nanofibers ............................................ 32 
3.2. 2. Synthesis of N-methacryloyl L-histidine methylester (MAH) ...................... 33 
3.2. 3. Synthesis of MAH-Cu(II) Metal-chelate Monomer ...................................... 33 
3.2. 4. Preorganization of MAH-Cu(II) Monomer with Cyt c .................................. 33 
3.2. 5. Preparation of Cyt c-MIP Nanofibers .......................................................... 34 
3. 3. Batch Adsorption Studies .............................................................................. 35 
3.3. 1. Rebinding Experiments .............................................................................. 35 
3.3. 2. Selectivity Studies ...................................................................................... 35 
3. 4. Fabrication of QCM Nanosensors ................................................................. 36 
3.4. 1. Surface Characterization of QCM Nanosensors ........................................ 37 
3.4. 2. Kinetic Studies with QCM Nanosensors ..................................................... 37 
3.4. 3. Selectivity Studies of QCM Nanosensors ................................................... 37 



 vii 

4. RESULTS AND DISCUSSION ....................................................................... 38 
4. 1. Preparation of Surface Imprinted MIP Nanofibers ......................................... 38 
4.1.1. Synthesis of MAH-Cu(II) Complex ...................................................... 38 
4.1.2. Characterization of Cyt c-MIP nanofibers .................................................... 39 
4. 2.  Study of Conditions of Polymerization .......................................................... 42 
4.2. 1. Effect of Monomer/Template Ratio ............................................................. 43 
4.2. 2. Effect of Total Monomer Ratio .................................................................... 44 
4.2. 3. Effect of Polymerization Time ..................................................................... 45 
4. 3. Rebinding studies .......................................................................................... 46 
4.3. 1. Effect of pH ................................................................................................. 46 
4.3. 2. Effect of Temperature ................................................................................. 47 
4.3. 3. Effect of NaCl Concentration ...................................................................... 48 
4. 4.  Binding Isotherms ......................................................................................... 49 
4. 5. Binding Kinetics ............................................................................................. 53 
4. 6. Thermodynamic Analyses ............................................................................. 58 
4. 7. Selectivity Studies ......................................................................................... 59 
4. 8. QCM Studies ................................................................................................. 61 
4.8. 1. Surface Morphology of QCM nanosensor .................................................. 62 
4.8. 2. Kinetic Studies with Cyt c-MIP QCM nanosensor ...................................... 63 
4.8. 3. Mathematical analysis of Cyt c-MIP QCM sensor data .............................. 65 
4.8. 4. Equilibrium isotherm models ...................................................................... 66 
4.8. 5. Selectivity studies ....................................................................................... 68 
4.8. 6. Reproducibility ............................................................................................ 70 
5. CONCLUSION ................................................................................................ 71 
REFERENCES ...................................................................................................... 75 
APPENDIX ............................................................................................................ 89 
CURRICULUM VITAE ........................................................................................... 92 
 
 
 
 

 

 

 

 

 

 

 

 
 



 viii 

FIGURES 

Figure 2. 1. Representation of molecular imprinting process. ................................. 5 

Figure 2.2. The schematic representation of complexation of TC/Fe2+/MAA. ......... 9 

Figure 2.3. The binding interactions of MIP via a.) H-bonding and b.) Metal ion 

coordination. .......................................................................................................... 10 

Figure 2.4. Metal coordination between metal ion (Me) and a.) TED and b.) IDA. 11 

Figure 2.5. Schematic representation of binding of various bis(imidazole) 

molecules.. ............................................................................................................ 12 

Figure 2.6. The schematic presentation of protein imprinting process with metal ion 

coordination. .......................................................................................................... 13 

Figure 2.7. The representation of MAH-Cu(II)-L-histidine formation. .................... 14 

Figure 2.8. Schematic representation of preparation of surface imprinted 

nanowires. ............................................................................................................. 16 

Figure 2.9. Schematic representation of preparation of surface imprinted silica 

nanoparticles. ........................................................................................................ 17 

Figure 2.10. Mass transfer mechanisms through bead (A) and membrane (B) .... 19 

Figure 2.11. Schematic presentation of accessible binding sites of bulk and 

nanosized MIPs.. ................................................................................................... 19 

Figure 2.12. SEM images of nanofibers having different morphologies. Beaded (a), 

non-porous (b), core-shell (c), porous (d). ............................................................. 20 

Figure 2.13. SEM images of PVA-PE nanofibers (A), Cibacron Blue F3GA-

attached PVA-PE nanofibers. ................................................................................ 22 

Figure 2.14. Schematic representation of preparation of electrospun MIP 

nanofibers using electrospinning equipments. ...................................................... 23 

Figure 2.15. SEM images of polypyrole nanowires. .............................................. 23 

Figure 2.16. Schematic presentation of preparation strategy of MIP nanotube 

membrane. ............................................................................................................ 24 

Figure 2.17. Schematic presentation of fabrication of MIP-PDA nanowires. ......... 25 

Figure 2.18. Schematic presentation of MIP-NPs on the SEM image of PET 

nanofibers. ............................................................................................................. 25 

Figure 2.19. TEM image of bacterial cellulose ribbon produced by the bacteria. .. 26 

Figure 2.20. SEM image of bacterial cellulose network containing bacteria cells.. 26 

Figure 2.21. Chemical structure of cellulose. ........................................................ 27 



 ix 

Figure 2.22. Schematic representation of QCM sensor chip ................................. 30 

Figure 2.23. Schematic view of the experimental setup for humidity sensor of QCM 

with BC nanofibers. ............................................................................................... 31 

Figure 3. 1.  Schematic presentation of preparation strategy of Cyt c-MIP QCM 

nanosensors. ......................................................................................................... 36 

Figure 4. 1. UV-vis spectra of MAH and MAH-Cu(II) complexes. ......................... 39 

Figure 4. 2. ATR-FTIR spectrum of MAH monomer. ............................................. 40 

Figure 4. 3. ATR-FTIR spectrum of BC. ................................................................ 40 

Figure 4. 4. ATR-FTIR spectrum of Cyt c-MIP nanofibers. ................................... 41 

Figure 4. 5. SEM image of Cyt c-MIP nanofibers. ................................................. 42 

Figure 4. 6. Contact angle measurment of Cyt c-MIP nanofiber. .......................... 42 

Figure 4. 7. Adorption capacities of the MIP nanofibers with different 

monomer/template ratio. ....................................................................................... 44 

Figure 4. 8. Adsorption capacities of MIP nanofibers with different weight ratio of 

total monomer.. ..................................................................................................... 45 

Figure 4. 9.  Adsorption capacities of MIP nanofibers with different polymerization 

time.. ...................................................................................................................... 46 

Figure 4. 10. Effect of pH of adsorption solution on adsorption capacity of MIP 

nanofibers. ............................................................................................................. 47 

Figure 4. 11. Effect of temperature of adsorption solution on adsorption capacity of 

MIP nanofibers. ..................................................................................................... 48 

Figure 4. 12. Effect of NaCl concentration on adsorption capacity of MIP 

nanofibers. ............................................................................................................. 49 

Figure 4. 13. Effect of equilibrium concentration Cyt c on adsorption capacity of 

MIP nanofibers. ..................................................................................................... 50 

Figure 4. 14. Langmuir isotherm of MIP nanofibers. ............................................. 51 

Figure 4. 15. Freundlich isotherm of MIP nanofibers. ........................................... 51 

Figure 4. 16. Binding kinetics of MIP nanofibers. .................................................. 54 

Figure 4. 17. First-order kinetics of adsorption of MIP and NIP nanofibers. .......... 56 

Figure 4. 18. Second-order kinetics of adsorption of MIP and NIP nanofibers. .... 56 

Figure 4. 19. Intraparticle diffusion kinetic model of MIP and NIP nanofibers. ...... 57 

Figure 4. 20. Effect of metal ion coordination and molecular imprinting process on 

adsorption capacity of Cyt c. ................................................................................. 60 



 x 

Figure 4. 21. The adsorption capacity of cyt c and competing proteins on Cyt c-

MIP nanofibers. ..................................................................................................... 61 

Figure 4. 22. AFM images of MIP nanofiber QCM chip. ........................................ 62 

Figure 4. 23. Dynamic response of Cyt c-MIP nanofibers QCM sensors with 

respect to frequency change. ................................................................................ 63 

Figure 4. 24. Dynamic response of Cyt c-MIP nanofibers QCM sensors with 

respect to mass change. ....................................................................................... 64 

Figure 4. 25. The relation between mass shift and cytochtome c concentration. .. 64 

Figure 4. 26. Determination of equilibrium analysis (Scathard). ............................ 66 

Figure 4. 27. Langmuir adsorption model. ............................................................. 67 

Figure 4. 28.  Freundlich adsorption isotherm. ...................................................... 68 

Figure 4. 29.  Langmuir-Freundlich adsorption isotherm. ...................................... 68 

Figure 4. 30. Selectivity of Cyt c-MIP QCM sensors. ............................................ 69 

Figure 4.29. Reproducibility of Cyt c-MIP QCM sensors. ...................................... 70 

 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 



 xi 

TABLES 

Table 4. 1. Langmuir and Freundlich parameters of MIP nanofibers .................... 52	
  

Table 4. 2. Comparison of Cyt c adsorption capacities of different adsorbents with 

MIP nanofibers prepared in this study ................................................................... 52	
  

Table 4. 3. Constants for first and second order kinetic and intraparticle diffusion 

kinetic model of adsorption of MIP and NIP nanofibers ........................................ 57	
  

Table 4. 4. Thermodynamic parameters of MIP nanofibers .................................. 58	
  

Table 4. 6. Kinetic parameters. ............................................................................. 65	
  

Table 4. 7. The equilibrium isotherms ................................................................... 66	
  

Table 4. 8. The parameters of equilibrium isotherms. ........................................... 67	
  

Table 4. 9. Selectivity of Cyt c-MIP QCM nanosensors to Cyt c and non-template 

proteins. ................................................................................................................. 70	
  

 
 
 



 xii 

SYMBOLS AND ABBREVIATIONS 

Symbols 

Q    Adsorption capacity 

 

 

Abbreviation 
 

MIP    Molecularly Imprinted Polymer 

Cyt c    Cytochrome c 

MAH    N-Methacryloyl-L-Histidine Methyl Ester 

MAH-Cu(II)   The complex of MAH monomer and cooper(II) ion 

MAH-Cu(II)-Cyt c  The complex of MAH monomer, copper(II) ion and Cyt c  

Cyt c-MIP   Cytochrome c Imprinted Bacterial Cellulose Nanofibers 

UV-Vis   Ultraviolet-Visible Spectroscopy 

SEM    Scanning Electron Microscopy 

AFM    Atomic Force Microscopy 

QCM    Quartz Crystal Microbalance 

Cyt c MIP QCM  Cytochrome c Imprinted Quartz Crystal Microbalance Sensor 

 

 

 

 

 

 

 

 

 

 

 

 

 



 1 

1. INTRODUCTION 

Purification, separation and isolation of proteins are necessary for further 

developments of bioscience and biotechnology. The purification of proteins is 

crucial since they posses therapeutic value for diagnosis and treatment.  The 

development of production processes of highly specific materials available to 

capture proteins requires rapid development of purification systems [1]. There is a 

growing interest in the development of purification processes to obtain materials 

with high specificity and capacity. In addition the purification process should be 

easy-to-apply and cost effective [2]. 

Molecular recognition refers to the affinity between two or more molecules via 

various types of interactions. Molecular imprinting is promising alternative to create 

highly selective binding sites through polymeric materials via molecular recognition 

[3]. There are many molecular imprinted polymers (MIPs), which show unique 

characteristics of this method targeting various types of molecules [4]. The 

molecule of interest namely a template molecule is surrounded by functional 

monomers and cross-linkers via covalent, non-covalent or metal ion coordination 

interactions. After the polymerization takes place, template molecule is removed 

from this polymeric structure leaving specific binding cavity. Surface imprinting is a 

widely used approach to improve the performance of MIPs by solving the 

problems of mass transfer limitations and removal of template molecules generally 

related with traditional molecular imprinting technique [5]. This method is 

especially significant for imprinting of macromolecules like proteins since mass 

transfer limitations for these large molecules is an important factor. The creation of 

thin films onto solid supports is one of the common approaches of surface 

imprinting method. This strategy enhances rebinding kinetics significantly because 

of the easy transportation of molecules through the binding sites on the or near the 

surface of the material. Other important advantage of this method is simple 

removal of target molecules leaving large amount of recognition sites onto the 

solid support. These features are especially significant for macromolecules like 

proteins. The purification of proteins using this type of materials which have thin 

films created onto the solid support is feasible regarding to important features 

explained above [6], [7], [8], [9]. Molecular imprinting via metal ion coordination 

provides high selectivity and stability. In this context metal ions play role as a 



 2 

mediator between template protein and monomer contributing the well-defined 

orientation of this complex. The utilization of metal ion coordination during 

molecular imprinting favors the preparation of polymerization process in aqueous 

environment [10].  

Protein purification is generally performed with traditional chromatography, which 

is packed with beads. In order to avoid the drawbacks of packed beds such as 

high pressure loss and mass transfer limitations, nanofiber membranes bearing 

high surface area are powerful alternatives. Nanofibers are typically prepared via 

electrospinning technology and used in many application areas. Bacterial cellulose 

(BC) is a biocompatible, thermal and chemically stable material, which is produced 

by A. xylinum bacteria with the presence of only carbon source [11]. Also it is 

prepared easily without requiring any special device. BC has high purity when 

synthesized and it does not need any further purification steps after the removal of 

bacteria cells from the membrane [12]. 

Cytochrome c is an essential protein for the transportation of electron in 

respiratory chain of the cell. It is a small and highly stable protein. In recent years, 

its role in apoptosis process was investigated and it was reported as a therapeutic 

protein.  

In this thesis, surface imprinted cytochrome c nanofibers were prepared onto the 

surface of bacterial cellulose nanofibers via metal coordination interactions (Cyt c-

MIP nanofibers) and used for efficient separation cytochrome c molecules from 

aqueous solutions. Firstly, N-methacryloyl-L-histidine methylester (MAH) as a 

metal chelate monomer was synthesized by reaction of methacyloyl chloride and 

histidine. Then it was preorganized with copper ions to obtain metal chelate 

monomer, MAH-Cu(II). The ternary complex was prepared with the addition of Cyt 

c molecules bearing one surface histidine to this MAH-Cu(II) complex and allowed 

to interact to form stable structure. After polymerization with cross-linker, template 

molecules were extracted from polymeric structure. The characterization of Cyt c-

MIP nanofibers was performed with ATR-FTIR, SEM and contact angle 

measurements. To determine the optimum conditions of polymerization process, 

the effects of monomer/template ratio, total monomer ratio and polymerization time 

were investigated. The adsorption properties of Cyt c-MIP nanofibers were 

evaluated at different experimental conditions in batch wise system from aqueous 

solutions. The desorption was achieved with 1 M NaCl solution The suitable 



 3 

binding equilibrium isotherm was determined as Langmiur isotherm model. 

Selectivity properties of Cyt c-MIP nanofibers were investigated using bovine 

serum albumin, hemoglobin, myoglobin and lysozyme as non-template proteins. 

The binding characteristics of the surface imprinted nanofibers were also 

examined with QCM sensor studies. QCM nanosensors were prepared via casting 

Cyt c-MIP and NIP nanofibers onto the chip surface. The results obtained from 

QCM studies were in conformity with the results of batch rebinding studies.  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



 4 

2. GENERAL INFORMATION 

Protein purification processes has been performed for more than 200 years with 

various kinds of processes such as precipitation, centrifugation, ion-exchange 

chromatography, affinity chromatography etc. [13].  The purification of proteins is 

an attractive field since most proteins have therapeutic value regarding to 

pharmaceutical industry. In order to purify the protein of interest from its mixture, 

which may contain several hundreds of other proteins, the recognition mechanism 

between protein and adsorbent material is necessary to develope. Molecular 

recognition based on lock and key model is a basic process found in nature. It 

bears affinity interactions between two or more molecules. Molecular imprinting is 

based on the molecular recognition process is a promising alternative for protein 

purification with high selectivity [14].   

2.1. Molecular Imprinting Process 

Molecular imprinting is a technology creating highly specific recognition sites into 

cross-linked polymer using template molecule as a target. The molecule of interest 

is allowed to interact with functional monomers either by covalent, non-covalent or 

metal ion coordination interactions. This template molecule(s)-monomer(s) 

complex is then preserved via polymerization to create a binding site recognizing 

the template molecule. After extraction of template molecule from the polymeric 

material, recognition cavity complementary to template molecule with both 

functionality and shape is formed (Figure 2.1) [15], [16]. The molecular imprinted 

polymers (MIPs) are resistant to elevated temperature and pressure, inert to 

chemicals, stable and cheap [17]. 

                                  



 5 

Figure 2. 1. Representation of molecular imprinting process. a.) Pre-polymerization 

complex of template and functional monomers (red, green and brown) via 

covalent, non-covalent and metal ion coordination interactions. b.) Polymerization 

process with cross-linkers. c.) Template (yellow) removal with extraction solvents 

to create specific binding sites complementary to template molecule Reprinted 

from ref. [18]. 

 

2.1. 1. Factors Affecting The Molecular Imprinting Process 

The design of molecular imprinting is a big challenge since the structure depends 

on many variables such as chemical structure and amount of template, functional 

monomer, cross linker, solvent, initiator, method of initiation and temperature [19]. 

Binding characteristics, stability, selectivity and binding kinetics are governed by 

recognition cavity formed into polymeric structure. In order to obtain high 

recognition capacity with high selectivity, it is important to optimize these 

parameters that are explained below [20]. 

2.1. 2. Template 

Molecular imprinting method is applied to various kinds of templates such as metal 

ions, antibiotics, drugs, sugars, amino acids, peptides, proteins and even cells 

[21], [22], [23], [24], [25]. The nature of the template will determine the choice of 

the functional monomers. In order to create a well-defined orientation between 

functional monomers and template molecule, target should contain functional 

groups able to interact with monomers. Also it should be stable in the course of 

polymerization process. In order to provide high selectivity, stability of template is 

essential to create high fidelity for recognition cavity resulting minimum change of 

conformational state after rebinding to binding site [26]. 

2.1. 3. Functional Monomer 

Chemical structure of functional monomers is also an issue of major importance 

for efficient imprinting process since they should interact with template molecule in 

the right orientation to preserve conformational state of the target [27]. The most 

frequently used functional monomers are acrylic acid, methacrylic acid, 

trifluoromethacrylic acid, 4-vinylbenzoic acid, styrene, acrylamide and methyl 



 6 

methacrylate. The ideal monomer for molecular imprinting is selected due to the 

strength and chemical structure of the interactions between monomer and 

template molecules [28]. 

2.1. 4. Cross-linker 

The cross-linkers also serve important role for recognition capability of MIPs 

providing rigidity of polymer network that is necessary to stabilize the recognition 

sites [29]. The most common cross-linkers are ethylene glycol dimethacrylate, 

divinylbenzene, N,N-ethylenebismethacrylamide and trimethylolpropane 

trimethacrylate. Ideally cross-linkers should not interact with template molecules to 

avoid non-specific adsorption [30]. 

2.1. 5. Initiation Method and Temperature 

The formation of free radicals is widely used technique for initiation of 

polymerization of MIP synthesis and usually carried out by thermally and 

photolytically. Azonitriles are commonly used initiators, which are decomposed by 

heat or UV light. The ideal initiator should control polymerization degree that is 

exothermic reaction. The MIPs prepared using photochemical or thermal initiation 

techniques was utilized for evaluation of enantioselectivity properties. It was found 

that photolytic method at low temperature achieved better enantioselectivity [31]. 

Monomer-template complexation is equilibrium-based reaction and depends on 

temperature. Temperature effect for chiral recognition was investigated using 

azobisnitriles as initiators with thermal and photolytic methods. It was reported that 

lower temperatures achieve better enantioseparation [32].  

2.1. 6. Polymerization Time 

Polymer morphology is directly depended on polymerization time. When it is long, 

polymer becomes more rigid in structure since prolonged polymerization period 

allows consuming all the polymerizable double bonds. This may provide defined 

binding cavities resulting higher selectivity properties. Also it may cause to obtain 

slow binding kinetics since transport properties of rigid materials are limited. On 

the other hand, when polymerization time is short, polymer structure becomes 

loose. It was shown that separation factor increases with increasing polymerization 



 7 

time when the thermal and UV light source (0.016 W/cm2) used as an initiation 

method [33], [34]. 

2.1. 7. Solvent 

Polymer morphology and imprinting process is significantly affected by nature and 

amount of solvent used in the polymerization procedure. Solvent solubilizes the 

components of polymerization syrup and provides the stabilization of template-

monomer complex, which is important step for imprinting process. Furthermore the 

porosity of the MIP prepared is strongly depended on solvent, which serves as a 

porogen. The utilization of appropriate solvent provides the fabrication of high 

specific surface area that is necessary for high adsorption capacity [35]. 

2. 1. Types of Molecular Imprinting  

The driving forces required for the interaction between template and functional 

monomer are covalent bonds, non-covalent bonds and metal ion coordination [36]. 

Molecular imprinting methods prepared using these interactions are explained 

below. 

2.2. 1. Covalent Imprinting 

In this type of molecular imprinting procedure, template molecule is copolymerized 

with functional monomers. After polymerization, covalent bond is cleaved with 

chemicals leading a fixed cavity with well chemical orientation. The substantial 

advantage is to create exact-fit recognition sites reducing non-specific interactions 

and preventing leakage of template molecules because of the formation of stable 

covalent bonds. The major disadvantage is to use harsh chemicals to chemically 

cleave the template molecules resulting the possible damage to the imprinted sites 

[37]. Since cholesterol-imprinted polymers prepared via covalent imprinting 

method were found to have higher chromatographic separation efficiency of 

cholesterol than cholesterol-imprinted polymers prepared via non-covalent 

imprinting method resulting reduced peak broadening and tailing [38]. However 

covalent bonding gives strong interaction between template and monomer, it has 

slow rebinding kinetics that is an important issue for rapid separation and 

purification processes and low template removal. 

 



 8 

2.2. 2. Non-covalent Imprinting 

To synthesize MIPs via non-covalent imprinting approach, template and functional 

monomers were pre-organized before polymerization using secondary interactions 

such as H-bonding, van der Waals interactions and Coulomb forces. The major 

advantage of non-covalent imprinting is the ability to apply this method for large 

variety of template molecules. Also it is a direct, easy-to-apply and flexible method. 

The major disadvantages are the weak complexation of template and monomers 

and also the formation of heterogeneous binding sites when used excess of 

functional monomer [39]. Despite water is common solvent for molecular 

imprinting since many biomolecules have limited solubility in organic media; the 

recognition capacity is low due to weakness of hydrogen bonds. Also imprinting 

effect may be weakened in aqueous environment since polar solvents compete 

with hydrogen bonding interactions [40]. 

2.2. 3. Metal Ion Mediated Molecular Imprinting 

Metal ion coordination with biological molecules is well suited to molecular 

recognition due to its specificity and stability. In metal ion coordination during 

imprinting process, metal chelating monomers are pre-organized to metal ion, 

generally a transition metal ion, which, in turn, coordinates the template molecule. 

Metal ions are employed as mediator that directs functional monomer and 

template molecule to establish a high fidelity of imprint with high specificity [41], 

[42], [43]. Metal ion coordination has higher strength with respect to hydrogen 

bonding which makes it more stable in water for example, binding energy of the 

complex of Cu2+ and imidazole residue of histidine is 4.8 kcal/mol and it is more 

than 1 kcal/mol for typical hydrogen bonding interaction [44]. Additionally metal ion 

coordination is a fast binding process and binding strength can be adjusted by 

choosing appropriate metal ion for a defined template molecule. Furthermore it is 

possible to replace the metal ion with another one to enhance the selectivity or use 

the MIP for different aim [45], [46], [47]. Therefore within this context, metal ion 

coordination approach has an important potential for preparation of highly specific 

MIPs in aqueous medium. 

The most important step of the preparation of the MIP is the pre-arrangement of 

the functional monomer, metal ion and template molecule. This ternary complex is 

then polymerized with cross-linking agents initiated thermally or by UV light. After 



 9 

polymerization the template molecule is easily removed with appropriate 

chemicals. In order to use this resultant material with other metal ions, it can be 

washed with complexing agents such as EDTA to remove all the metal content 

and then reload the other metal ion [48]. 

2.2.3.  1. Key Parameters for Metal Ion Coordination 

The selection of metal ion is one of the most important parameter to create 

specific recognition. Template molecule dominates selection of the type of the 

metal ion used for imprinting process. The type of the metal ion defines binding 

strength of the template-metal ion-monomer complex and the spatial arrangement 

of this complexation. In order to obtain high specificity, coordination mode of the 

metal ion-monomer complex should be determined to guarantee the 

immobilization of the metal ions in the imprinted matrix. The molecularly imprinted 

polymers for extraction of tetracyclines -a large family of common antibiotics- from 

biological fluids was prepared in aqueous medium utilizing Fe2+ as mediator and 

methacrylic acid (MAA) as functional monomer (Figure 2.2) [49]. Different metal 

ions such as Mg2+, Fe2+ and Cu2+ were complexed with tetracycline (TC) in the 

preparation of MIP. It was found that Fe2+ could obtain high recognition capacity 

due to specific coordination interaction between TC and MAA. 

                           

                                 
Figure 2.2. The schematic representation of complexation of TC/Fe2+/MAA. 

Reproduced from ref. [50]. 



 10 

As mentioned before, the self-assembling of template and monomers is the pre-

polymerization step for the fabrication of MIP. Utilizing metal ion as mediator 

assembles a bridge between template and monomer through coordination bond. 

(S)-naproxen was complexed with 4-vinylpyridine through coordination with Co2+ 

and thus MIP with high selectivity was prepared [47]. Figure 2.3 shows the 

schematic presentation of this complex and further the binding of template and 

monomer via hydrogen bonding. It was shown that the absence of metal ion, 

which means in that case using hydrogen-bonding interactions, resulted reduced 

selectivity. The stoichiometry of the complex used was determined with UV 

spectrum by titrating monomer and template. The optimization of monomer 

amount is necessary for the design of MIPs. High amount of monomer results low 

adsorption capacity due to inefficient template removal and low amount of 

monomer causes incomplete organization of template and monomer. In the same 

manner, the amount of metal ion plays important role to enhance selectivity. In 

order to form highly specific recognition sites complementary to template, the 

stoichiometric amount of metal ion to bridge the specific interaction between 

template and monomer with high stability is necessary [51]. 

                             
Figure 2.3. The binding interactions of MIP via a.) H-bonding and b.) Metal ion 

coordination. Reprinted from ref. [52]. 

 

The other parameter is the influence of anion used since it may participate in the 

recognition process. Molecularly imprinted solid phase microextraction fiber was 

developed to recognize thiabendazole (TBZ) - a kind of fungicide - via the metal 

coordination interaction. It was found that enrichment properties in aqueous 

solutions were improved with respect to hydrogen bonding interactions. Four 



 11 

different copper salts as acetate, sulfate, nitrate and chloride were evaluated with 

respect to adsorption capacity and copper (II) acetate was found to have the 

highest adsorption capacity. This demonstrates the effects of anion to the 

recognition process since it changes the size and shape of the template-metal-

monomer complex [53].  

2.2.3.  2. Metal Ion Coordination for Bis-imidazole Recognition 

Metal ion coordination with its specificity and stability is well-suited procedure for 

molecular recognition of biological molecules which is exemplified by the 

chromatographic method namely IMAC (Immobilized Metal Affinity 

Chromatography) [54]. It has been developed by Porath and protein purification 

was achieved via binding of electron donor groups on protein surface and metal 

ion immobilized on the support surface [55]. The amino and carboxyl groups of 

amino acid participate in the fabrication of metal-amino acid complex. Especially 

histidine containing peptides form stable complex with metal ions due to metal 

coordination between metal ion and imidazole side chain of amino acid. The 

common chelating ligands are iminodiacetic acid (IDA), nitrilotriacetic acid (NTA) a   

nd tris carboxymethyl ethylene-diamine (TED) and are shown below (Figure 2. 4). 

                        

          

                  a.)                                                               b.) 

Figure 2.4. Metal coordination between metal ion (Me) and a.) TED and b.) IDA. 

Reproduced from ref. [56]. 

 
The complementarity between metal ion and template molecule forms specific 

assembly prior to polymerization. After polymerization when template is removed 

from polymer matrix, there exists a very specific binding cavity complementary to 

template due to this specific arrangement of functional groups around template 



 12 

molecule. In order to prepare selective abiotic receptors for 1,4-Bis (imidazol-1-

ylmethyl) benzene (2 in Figure 2.5) which is analog of surface histidine bearing 

proteins, metal chelating monomer Cu(II)-(N-(4-vinylbenzyl)-imino)diacetic acid 

was pre-organized with template molecule and then polymerized in the presence 

of cross linking agent [57].  The affinity for molecule 2 was higher than the analog 

molecules 4,4’-Bis(imidazol-l-ylmethyl) biphenyl (4) and 1-Imidazol-l-ylmethyl)-4-

(pyrrol-l-ylmethyl) benzene (6) which contain single imidazole. This bigger affinity 

may be explained by two point binding mechanism of the template to MIP and it 

shows that the importance of templating is for distribution of metal ions through the 

polymer matrix. 

                               
Figure 2.5. Schematic representation of binding of various bis(imidazole) 

molecules. Reproduced from ref. [58]. 

 
2.2.3.  3. Metal Ion Coordination for Amino Acid, Peptide and Protein 
Recognition  

Amino acid imprinted polymers were prepared via metal ion coordination for chiral 

separation of various amino acids from aqueous solutions [40].  MIPs were 

prepared with the complex of Cu(II)-N-(4-vinylbenzyl)iminodiacetic acid as a metal 

chelate monomer and amino acid as a template in the presence of cross linkers. 

All the MIPs showed high enantioselectivity for the template and also it was 



 13 

reported that enantioselectivity depends on both size and shape of the side chain 

of amino acid residue. 

Hochuli et al. has been firstly developed a new quadridentate chelating ligand 

nitrilotriacetic acid (NTA) for protein purification via metal chelate chromatography 

[59]. It was reported that NTA forms more stable coordination interaction with both 

Cu(II) and Ni(II) than the interaction of IDA with each of these metals. NTA when 

complexed with Ni(II) occupies four positions in metal octahedral coordination 

sphere and leaving two for dipeptide His-Ala interaction [60]. It was achieved 

selective polymeric receptors to separate between peptide sequence via strong 

coordination NTA, Ni and His-Ala.  

Kempe et al. was first developed metal mediated molecular imprinting procedure 

for protein imprinting [61]. Molecular imprinted polymers were prepared to 

recognize ribonuclease A (RNase A) through metal coordination utilizing N-(4-

vinyl)-benzyl iminodiacetic acid (VBIDA) as the metal chelating monomer and 

copper ion as the mediator in the presence of the protein onto methacrylate-

derivatized silica particles (Figure 2.6). There has been growing attention in metal 

mediated protein imprinted polymers [62], [63], [64], [65]. All these studies shows 

promising properties of metal coordination for the preparation of highly selective 

MIPs via stable and specific arrangement of metal ions with protein surface for 

protein separation and recognition. 

                            
Figure 2.6. The schematic presentation of protein imprinting process with metal ion 

coordination. Reprinted from ref. [66]. 



 14 

A thermoresponsive macroporous hydrogel for lysozyme recognition was 

developed via molecular imprinting method based on metal ion coordination 

between protein and metal ion [62]. N-(4-vinyl)-benzyl iminodiacetic acid (VBIDA) 

as the metal chelate monomer was pre-organized with Cu(II) as the mediator and 

lysozyme as the template and then polymerized with N-isopropylacrylamide 

(NIPAAm) for thermoresponsiveness, acrylamide for mechanical strength and 

N,N-methylenebisacrylamide for cross-linking. The Lysozyme-MIP prepared via 

metal ion coordination between VBIDA, Cu(II) and lysozyme demonstrated higher 

protein recognition than Lysozyme-MIP prepared via electrostatic interactions 

obtained with pre-organization of VBIDA and protein. Also it was confirmed with 

selectivity tests that metal ion coordination was important for appropriate 

positioning of the binding groups around the template molecule.  

L-histidine imprinted polymers were developed via metal coordination between 

Cu(II) as the mediator, L-histidine as the template and N-methacryoyl-(L)-histidine 

(MAH) as the metal chelating monomer for selective separation of cytochrome c  

(Cyt c) (Figure 2.7) [65]. The chromatographic separation of cytochrome c and 

ribonuclease A was also achieved.  

                                   
Figure 2.7. The representation of MAH-Cu(II)-L-histidine formation. Reprinted from 

ref. [67]. 

 

2. 2. Protein Imprinting 

Specific molecular recognition, which is coordinated primarily by proteins, is the 

basic phenomenon that controls all the biological processes. The recognition of 



 15 

proteins with high affinity and selectivity is a big challenge since natural 

biomacromolecular receptors are not stable for long-term processes and also 

require high costs. Thus production of artificial recognition elements, which mimic 

the natural counterparts with high selectivity and capacity, is a great demand 

requiring cost-effective, robust and reusable alternatives. Synthesis of molecularly 

imprinted polymers capable of selective recognition of proteins offers great 

potential providing low cost, easy to prepare and unique recognition properties 

[68]. 

There are some obstacles when imprinting proteins, which are largely absent 

when imprinting of small molecules and these are based on protein properties 

such as molecular size, complexity, conformational flexibility and solubility. 

Proteins have large molecular size resulting poor mass transport properties when 

binding through some MIP monolithic columns, which have dense polymeric 

structure [69]. Proteins have large number of functional groups over a large 

surface area. This complex structure should be considered when selecting 

appropriate functional monomers for protein imprinting process to avoid multiple 

weak interactions that favor non-specific binding. Also polymerization conditions 

may cause denaturation of proteins or may change the conformational states since 

proteins are flexible in nature [70]. Furthermore proteins are water-soluble 

compounds, which is not compatible with traditional MIP procedure since majority 

of molecular imprinting processes take place in organic media. The major 

strategies to synthesize MIPs for selective recognition of proteins are explained 

below.  

2.3. 1. Bulk Imprinting 

The principle of bulk imprinting of proteins is to recognize the shape via templating 

the protein into the polymeric matrix. There are different types of protein-imprinted 

matrices such as hydrogels, sol-gels, cryogels and monoliths that prepared via 

bulk imprinting [67], [71], [72], [73]. However bulk imprinting is simple and common 

method to prepare specific MIPs for protein recognition, the system has major 

limitation, which is poor accessibility of imprinted cavities. Since easy removal and 

fast rebinding of templates is the fundamental requirement of any system, the 

protein imprinting via bulk imprinting method is limited with the control of porosity 

and pore size of the matrices [5]. 



 16 

2.3. 2. Surface Imprinting 

Since imprinted sites are located at the surface or close to the surface of the 

support material, surface imprinting presents an important alternative to bulk 

imprinting by improving the mass transfer properties and enhancing template 

removal of MIPs. The surface imprinted polymers let free access for 

biomacromolecules such as proteins to binding sites by using specific areas of 

proteins as a target [74]. 

One of the common methods to synthesize surface imprinted polymers has two 

steps; firstly the template is immobilized onto the solid support and lastly the 

support is dissolved after polymerization with monomers and cross-linkers. This 

method was used to prepare different types of materials such as nanowires [75] 

(Figure 2.8) and porous silica beads [76], [77].  

 

 
 

Figure 2.8. Schematic representation of preparation of surface imprinted 

nanowires. Reprinted from ref. [78]. 

 
Nanoporous alumina membrane was used to prepare protein-imprinted nanowires. 

Firstly alumina membrane surface was modified with aldehyde to immobilize the 

protein onto surface, then the pores of the membrane was filled with the 

polymerization medium containing AAm and MBAAm. After the polymerization in 

the nanopores, the alumina membrane was cleaved resulting protein binding sites 

on the surface. To show the universality of the procedure, bovine serum albumin, 



 17 

bovine cytochrome c and horseradish peroxidase were used as template proteins. 

The surface imprinted nanowires demonstrated higher template protein recognition 

than the control nanowires for all of the template proteins used.  

The other common approach for surface imprinting of proteins is the fabrication of 

thin films on the suitable substrates. Different types of supports were used such as 

polypyrole and polystyrene for the preparation of sensing material and micro-

plates respectively [79], [80]. The template protein is attached onto support 

material via covalent bonding [81] or self-assembly method [82]. The Lys imprinted 

matrix was prepared via self-assembly of protein molecules onto vinyl modified 

silica nanoparticles (Figure 2.9). Then highly dilute mixture of functional monomers 

and cross-linkers (0.4 w%) were polymerized over the template protein in order to 

create very thin recognition film onto the silica nanoparticles. To investigate the 

recognition property of these surface-imprinted silica nanoparticles competitive 

adsorption experiments containing the template protein (lysozyme) and the 

competitor protein (cytochrome c) were performed. It was found that MIP 

nanoparticles showed high selectivity for the template lysozyme against 

cytochrome c.  

 

 
Figure 2.9. Schematic representation of preparation of surface imprinted silica 

nanoparticles. Reprinted from ref. [83]. 

 

2. 3. Affinity Nanofiber Membranes for Protein Recognition 

Protein purification is performed traditionally by using packed bed 

chromatography. However there are some limitations of this method such as high-

pressure loss, long operation time and difficult scaling up since mass transfer of 

protein molecules is carried out with diffusion through the micro channels of 



 18 

beads, which is packing material in the packed bed. Furthermore it may exist 

channeling caused by non-homogenous distribution of beads through the column 

resulting the decrement of column efficiency [84].  

Porous membranes are powerful alternatives to avoid these technical limitations of 

the packed beds. The most important advantage of the membrane 

chromatography is convection of proteins through the binding sites. Pressure loss 

of membranes is significantly lower than that of packed beds. The binding is 

generally independent of feed flow rate resulting ability to work with high working 

flow rates. The other advantage is the ability to scale up. The interaction between 

the protein and the material is performed through the interconnected pores of the 

membranes as can be seen at Figure 2.10. The convection is dominant mass 

transfer process through pores of membranes since molecules are transferred via 

diffusion through dead-ended pores of beads. The diffusional resistance is avoided 

when used membranes and thus the only mass transfer resistance becomes the 

film diffusion on the surface of the membrane. Mass transfer limitations are 

significantly lowered since film diffusion is much more faster process than pore 

diffusion. Despite the membranes are thinner than beads, pressure losses are 

lowered with the help of interconnected pores of the membranes and thus working 

flow rates and efficiency will be increased.  

 
A) Packed bed chromatography 

 
B) Membrane chromatography 

Bulk convection 

Film diffusion 

Pore diffusion 

Bulk convection 

Film diffusion 



 19 

Figure 2.10. Mass transfer mechanisms through bead (A) and membrane (B) 

Reprinted from ref. [85]. 

 
The development of materials for purification and recognition of proteins with high 

capacity and selectivity becomes important issue for the economical and efficient 

purification of valuable products after the developments of production of 

biotherapeautics. In the lights of development of nanotechnology, preparation of 

molecularly imprinted polymers in nanoscale has been gaining more attention 

since these nanostructures show superior properties compared with conventional 

macrostructures [86], [87]. Recently the preparation of nanosized molecularly 

imprinted polymers and their application areas have been reviewed elsewhere 

[88], [89], [90]. Figure 2.11 demonstrates the distribution of effective recognition 

sites of nanosized MIPs comparing with conventional MIPs. Nanosized MIPs 

perform better site accessibility thus resulting fast binding kinetics and higher 

rebinding capacity than traditional MIPs since all the templates can be easily 

removed from the surface of the nanosized matrix leaving binding sites ready for 

target molecules [91].  

 

         
Figure 2.11. Schematic presentation of accessible binding sites of bulk and 

nanosized MIPs. Reprinted from ref. [78]. 

 



 20 

Affinity matrices are materials having specific recognition binding sites between 

ligand and target molecules. Despite membranes perform fast binding kinetics and 

low operation time; the adsorption capacity of membranes may be low. To 

overcome this issue affinity nanofibers draw attention as remarkable alternatives. 

Affinity nanofiber chromatography is a promising method for protein purification 

having both high efficiency of nanofibers and selectivity of chromatographic 

materials [92]. Nanofibers having high surface area/volume ratio show great 

potential to utilize in the various application areas requiring porosity. 

Electrospinning is one of the most widely used techniques for the preparation of 

fibers having diameter of nanometer/micrometer scale. Electrospinning has many 

advantages such as simplicity of handling, ability to scale up and easy-to-apply to 

various polymeric structures. Electrospun nanofibers having 10-100 nm diameters 

can be utilized in various application areas such as tissue engineering, drug 

delivery, enzyme immobilization, removal of heavy metals, biosensors and 

production of reinforced composite materials [93], [94], [95], [96], [97]. Also using 

this method nanofibers having different morphologies such as beaded, porous, 

non-porous and core-shell can be prepared (Figure 2.12). 

                             
Figure 2.12. SEM images of nanofibers having different morphologies. Beaded (a), 

non-porous (b), core-shell (c), porous (d). Reprinted from ref. [98]. 

 
Nanofibers has been gaining much attention because of having higher surface 

area/volume ratio than microfibers. Surface area increases with decreasing of the 

fiber diameter. Novel electrospun carbon nanofibers modified with carboxylic acid 



 21 

having high capacity was prepared for protein adsorption. Fiber diameter of the 

electrospun carbon nanofibers was 300 nm since fiber diameters of conventional 

carbon microfibers is typically 10 µm. Increment of the surface area of the 

nanofibers by 30 fold enhanced protein adsorption capacity significantly [99].  

Specific purification of many molecules such as bisphenol A, glutamic acid, 

propranolol and herbicides is performed with porous and non-porous nanofibers 

[100-102], [102]. Non-porous matrices for protein purification providing fast binding 

kinetics and high adsorption capacity have been gaining much attention since 

porous materials having high diffusional mass transfer limitations. Thus diffusional 

resistances of small molecules like metal ions, amino acids and drug molecules 

and macromolecules like proteins can be reduced via the utilization of non-porous 

nanofibers [103]. The most important advantage of affinity adsorption with non-

porous nanofibers is the convection of fluid through pores of the nanofibers and 

high recognition of proteins on the surface of nanofibers. Non-porous electrospun 

nanofibers having these superior properties exhibits great potential for protein 

purification. 

Cellulose, polysulfone, polyacrylonitrile and nylon/chitosan electrospun nanofibers 

have been prepared recently [104], [105], [106]. Zhang and coworkers prepared 

ion-exchange support material modified with anion exchange ligand 

diethylaminoethyl (DEAE) using electrospun cellulose acetate nanofibers having 

diameters of 10 nm-1µm for protein purification [107]. DEAE-nanofibers exhibit 

higher albumin adsorption capacity compared with DEAE-cellulose microfibers and 

commercial DEAE-cellulose fibers. Zhu and coworkers have synthesized 

polyvinylalcohol-polyethylene nanofibers. They immobilized dye ligand namely 

Cibacron Blue F3GA that is cheap and chemically stable instead of antibodies that 

are expensive, requiring special conditions for storage and immobilization (Figure 

2.13). It was shown that Cibacron Blue F3GA-attached nanofibers exhibited higher 

albumin adsorption capacity compared with Cibacron blue F3GA-attached 

chitosan microbeads. 



 22 

    
Figure 2.13. SEM images of PVA-PE nanofibers (A), Cibacron Blue F3GA-

attached PVA-PE nanofibers. Reprinted from ref. [108]. 

 
Nanofibrous electrospun glycopolymers were also prepared with two 

glycopolymers containing cyclic poly (acrylonitrile-co-(α-allyl glucoside)) 

(PANCAG) and linear poly (acrylonitrile-co-(D-gluconamidoethyl methacrylate)) 

(PANCGAMA) performing high protein binding capacity with the help of the 

nanosized structure [109]. This type of materials having superior flow features and 

high permselectivity provide chiral separation from racemic mixtures.  

The novel molecularly imprinting procedure for the fabrication of selective binding 

sites directly by electrospinning containing the polymerization medium with 

template molecule, 2,4-dichlorophenoxyacetic acid which is a herbicide, was 

developed [101]. The polymerization mixture was electrospun onto the aluminum 

foil placed 20 cm from the capillary tip using experimental system shown in the 

Figure 2.14. Poly(ethylene terephthalate) (PET) was utilized as a supporting 

matrix. The recognition sites were fabricated via interaction between 

polyallylamine and template molecule. Molecularly imprinted nanofibers with high 

surface area was also prepared via electrospray deposition [110]. 

To obtain selective glutamic acid recognition, alumina membranes were utilized as 

a nanomold to synthesize MIP nanowires via template synthesis method [112]. 

Template molecule in that case glutamic acid, was covalently immobilized onto the 

walls of alumina membrane having pores of 100 nm diameter (Figure 2.15). After 

the polymerization using pyrole as a monomer, alumina membrane was removed 

via chemical dissolution method resulting polypyrole nanowires with glutamic acid 

recognition sites. High imprinting factor was achieved however binding kinetics 

was not so satisfactory since it reached equilibrium after 20 min. The same 



 23 

research group prepared magnetic MIP nanowires showing high specificity to 

template molecule and imprinting factor was approximately 6 using alumina 

membrane as a nanomold [113].  

 

 

Figure 2.14. Schematic representation of preparation of electrospun MIP 

nanofibers using electrospinning equipments. Reprinted from ref. [111]. 

 

                         

Figure 2.15. SEM images of polypyrole nanowires. Reprinted from ref. [50]. 

 
MIP nanotubes using anodic alumina oxide membrane with 100 nm pore diameter 

were prepared via ATRP, which is a kind of controlled radical polymerization 

technique [114]. ATRP initiators were immobilized onto the walls of alumina 

membrane and then membrane was immersed into polymerization medium 

containing template molecule (Figure 2.16). After removal of alumina membrane, it 



 24 

was achieved 11 fold higher rebinding capacity and 13 fold higher imprinting factor 

comparing with conventionally prepared bulk MIPs with MIP nanotubes with 

controlled size. 

 
a: Silanization with 3-aminopropyltrimethoxysilane 

b: Reaction with 2-Bromo-2-methylpropionyl bromide 

c: Polymerization of estradiol:4-vinylpyridine complex and ethylene glycol 

dimethacrylate 

d: Remove of estradiol 

Figure 2.16. Schematic presentation of preparation strategy of MIP nanotube 

membrane. Reprinted from ref. [115]. 

 
Silicon nanowires (SiNW) with unique mechanical properties, large surface area, 

high hydrophilicity and biocompatibility were used as a supporting matrix to obtain 

MIP coated silicon nanowires for fast and selective bovine hemoglobin recognition 

[116]. Thin PDA layer attached onto SiNW with a thickness of 10 nm was created 

with DA as a functional monomer (Figure 2.17). Template extraction after 

polymerization was found very high as %96.3 w/w since template molecules 

placed on the surface or near the surface of the nanowires. The imprinted 

nanowires showed high binding kinetics, it took 5 min to reach the 75% of the 

equilibrium. Molecularly imprinted nanowires achieving high specificity and fast 

binding kinetics was created.  

                 



 25 

Figure 2.17. Schematic presentation of fabrication of MIP-PDA nanowires. 

Reprinted from ref. [78]. 

 
Also the development of electrospun nanofibers embedded with molecularly 

imprinted nanoparticles is an promising approach to enhance surface area of the 

material [117], [118]. The theophylline and 17 β-estradiol imprinted nanoparticles 

200-300 nm in diameter were encapsulated onto PET nanofibers having 150 nm 

diameter (Figure 2.18).  

                  
Figure 2.18. Schematic presentation of MIP-NPs on the SEM image of PET 

nanofibers. Reprinted from ref. [119]. 

 

2. 4. Bacterial Cellulose 

Bacterial cellulose (BC) is produced by the genera Acetobacter, Rhizobium, 

Agrobacterium, and Sarcina. The most efficient producer is Acetobacter xylinum, 

which is a gram-negative acetic acid bacteria [120]. Bacterial cellulose is the 

primary metabolism product of the bacteria providing oxygen-rich surface and 

moisture for bacteria. Also it is considered that the pellicle protects the bacteria 

cell from the negative effects of UV light by surrounding it like a cage. The 

bacterial cellulose nanofiber ribbon with the producers bacteria cells is seen in 

Figure 2.19. The bacteria cell from its terminal complexes extrudes the linear 

glucan chains, which aggregate to form the nanofibers via twisting. These 

nanofibers then aggregate to form a ribbon possessing the diameter of 50-100 nm. 

Bacterial cellulose occurs at liquid-air interface with the inoculation of A.xylinum 

into the Hestrin-Schramm medium [121]. BC pellicles appear as a gelatinous 

material of variable thickness when floating on the surface of the medium. For its 

recovery, BC is firstly washed with distilled water to remove culture medium 



 26 

remaining in the pores of the membrane. Then BC is boiled in 1-3 w% NaOH 

solution to eliminate the cells and the components of the medium [122].  

 

 
Figure 2.19. TEM image of bacterial cellulose ribbon produced by the bacteria. 

Reprinted from ref. [123]. 

 

 
Figure 2.20. SEM image of bacterial cellulose network containing bacteria cells. 

Reprinted from ref. [124]. 



 27 

The chemical structure of bacterial cellulose is identical to plant cellulose and is 

shown in Figure 2.21. BC nanofibers are hundred times thinner than that of plant 

cellulose. Also bacterial cellulose has great purity avoiding lignin and 

hemicellulose. It has gained great attention since it possesses unique properties 

such as remarkable mechanical properties, high porosity, water uptake ratio, and 

biocompatibility. Also it can be molded in situ. Bacterial cellulose has many 

application areas including paper industry [125], food additive [126], metal removal 

from waste-water [127], antimicrobial filtration [128], enzyme immobilization [129], 

tissue engineering as a scaffold, vascular prosthetic device [130], [131], [132], skin 

tissue repair [133], and drug delivery [134].  

Bacterial cellulose nanofibers are prepared with high purity, however electrospun 

nanofibers are not pure after the synthesis. Since electrospun nanofibers are 

synthesized via polymerization processes, it is necessary to wash them with 

several times. This drawback of synthetic nanofibers makes bacterial cellulose 

nanofibers important alternative. Since BC nanofibers are non-porous nanofibers, 

they also demonstrate high capacity and high binding kinetics avoiding mass-

transfer limitations. Furthermore preparation of BC is simple since synthesis 

procedure does not require any special equipment. Thus the utilization of bacterial 

cellulose nanofibers for protein purification is advantageous providing unique 

properties of nanofibers without any necessity for any spinning device.  

 

           
Figure 2.21. Chemical structure of cellulose. 

 

2. 5. Cytochrome c 

Cytochrome c (Cyt c) is an essential heme containing protein found in plants, 

animals, and many unicellular organisms [135], [136]. Molecular weight is about 

12,000 daltons. It consists of a chain of about 100 amino acids as a primary 



 28 

structure however some higher orders organisms possess a chain of 104 amino 

acids [137]. The porphyrin ring of active heme center coordinates central Fe atom 

due to pyrole nitrogens and forms a square planar complex. Cyt c is modulating 

electron transfer in the mitochondrial respiratory chain due to the iron center 

changing ferric (Fe3+) and ferrous (Fe2+) state and generally regarded as a 

universal catalyst of respiration which makes it an efficient biological electron-

transporter [138]. It transfers electron between two large protein complexes called 

cytochrome bc1 complex and cytochrome c oxidase complex. It is a small and 

sphere like in shape [139]. The amino acid sequence of cytochrome c is highly 

conserved among different organisms since the genetic similarity of mouse and rat 

cytochrome c is 91% with the human cytochrome c [140]. Due to its small size and 

sequence homology, it can be used as a model protein in studies of cladistics, 

which is an approach of classification of biological organisms [141]. 

There has been many research articles investigating the role of cytochrome c for 

apoptosis process and identifying it as a mediator in apoptotic mechanism [142], 

[115], [143]. It was shown that Cyt c release from the cell has occurred within 1 h 

only after apoptosis starts resulting a fast and apoptosis-specific process [144]. 

Thus the extracellular cytochrome c is suitable as a biomarker of apoptosis.  There 

have been many articles reporting cytochrome c as a biochemical indicator for 

various diseases including cancer, liver damage and myocardial ischemia [145], 

[146], [147]. Thus screening of Cyt c may be useful as a clinical biomarker during 

cancer therapy [148]. Recently, there have been research articles investigating 

cytochrome c as a therapeutic protein. In this context, the approach for cancer 

therapy is to target cytochrome c as a therapeutic protein to cancer cells to initiate 

apoptotic process causing cell death [149], [150], [151]. These developments 

present cytochrome c as a valuable protein for medicine exhibiting great potential 

of being a biomarker of a disease and effective therapeutic choice.  

Furthermore cytochrome c is widely used in adsorption studies as a model protein 

since Cyt c is nearly spherical (molecular dimensions 2.6 × 3.2 × 3.3 nm3) in 

shape [152]. Also it was shown that cytochrome c as one of the hardest proteins 

due to adiabatic compressibility can be adsorbed onto solid support without any 

conformational change providing more reliable results than hemoglobin which is 

identified as a very soft protein [153], [154].  



 29 

The MIP hydrogel was prepared for lysozyme recognition and obtained successful 

results with an imprinting factor of 1.72 [155]. In that study MIP hydrogel for 

cytochrome c recognition was also synthesized and the adsorption isotherms of 

lysozyme and cytochrome c were compared. Cyt c has It was concluded that 

cytochrome c is a better choice as a template since it enhanced the adsorption 

capacity and the selectivity of the MIP due to its higher charge density locating on 

the surface of the protein leading more specific pattern for binding site creation. 

However lysozyme has low amount of surface charge causing formed recognition 

sites less specific.  

As mentioned before, the choice of template is one of the major steps that affect 

binding characteristics of MIPs. Thus it is an important parameter to determine for 

preparing selective adsorbents with high capacity. In conclusion the selection of 

cytochrome c as a model protein is suitable to utilize it as a template molecule to 

synthesize selective MIPs in this study.  

2. 6. QCM Sensors 

Over the past three decades, many developments have been taken place in the 

field of sensor technology. Since the biological sensing elements are not stable for 

long-term applications, synthetic receptors that mimic natural counterparts are 

desirable. In that field especially molecular imprinted biosensors present promising 

alternatives possessing extraordinary selectivity and sensitivity properties [156]. 

Molecular imprinting method provides preparation of highly stable and specific 

polymeric materials, which achieve selective recognition features with binding sites 

created complementary to template molecules in shape and size [157].  

After Sauerbrey discovered the dependency of quartz oscillation frequency with 

mass change on the crystal surface in 1959, the use of term quartz crystal 

microbalance (QCM) arises. Thus the linear relation between the change of mass 

on the crystal surface and change in frequency of the oscillating crystal at a 

specific frequency is shown by the Sauerbrey equation:  

∆m= -C ∆f 

This equation is valid for elastic surfaces such as thin adsorbed layers and it is not 

appropriate to use this equation for inelastic surface, which possess energy loss 

during oscillation. Therefore Sauerbrey equation becomes in valid when the mass 



 30 

change is bigger than 2% of the crystal mass [158]. There has been many 

publications related to QCM biosensors for rapid and real-time detection of metal 

ion [159], small molecules [160], [161], [162], amino acid [163],  protein [164] and 

even cell [165]. QCM sensor chip is shown schematically below. 

                           

                              
Figure 2.22. Schematic representation of QCM sensor chip Reprinted from ref. 

[166]. 

 
High surface area of sensing material is significant to improve the sensing 

properties of QCM sensors. Therefore nanomaterials such as nanoparticles, 

nanorods, nanowires and nanofibers have been received much attention since 

these nanostructures lead high sensitivity and fast response in sensor applications 

[167]. QCM sensors coated with bacterial cellulose were used for real-time 

detection of both humidity and formaldehyde [168], [169]. Highly stable, sensitive 

and cost-effective QCM sensors were fabricated via coating the crystal surface 

with bacterial cellulose nanofibers. The experimental setup was shown in the 

figure below. The results showed that bacterial cellulose with high surface area 

presented high sensitivity, good long-term stability and good linearity.  

 



 31 

              
Figure 2.23. Schematic view of the experimental setup for humidity sensor of QCM 

with BC nanofibers. Reprinted from ref. [169].  

 
Different types of sensor systems were used to detect cytochrome c such as 

Surface Plasmon Resonance (SPR). Alkyl thiols modified SPR sensors with 

different tail groups were evaluated for the detection of Cyt c [170]. The other one 

is Atomic Force Microscope (AFM) force spectroscopy to measure the force 

between protein and binding site [171]. 

In conclusion to prepare sensitive QCM sensor, bacterial cellulose nanofibers are 

great alternative to create selective recognition sites avoiding diffusional 

resistances especially for biomacromolecules.  

 

 

 

 

 

 

 

 

 

 

 



 32 

3.  EXPERIMENTAL METHOD 

3. 1. Materials 

Horse heart cytochrome c (Cyt c), chicken egg white lysozyme (Lys), bovine 

myoglobin (Myo), bovine serum albumin (BSA), bovine hemoglobin (Hb), N,N’-

Methylenebisacrylamide (MBAAm), L-histidine methylester dihydrochloride and 

methacryloyl chloride were all purchased from Sigma (St. Louis, USA). Radical 

initiator azobisisobutyronitrile (AIBN) was supplied by Fluka (Switzerland). 

Acetobacter xylinum (ATCC10245) was supplied from Agricultural Research 

Service Culture Collection (ARS, USA) in lyophilized form. Growth medium 

components, D-glucose, peptone, yeast extract, K2HPO4 and KH2PO4 were 

obtained from Merck (Darmstadt, Germany) in analytical grade. All other 

chemicals used were purchased from Merck A.G. (Darmstadt, Germany) unless 

otherwise noted. The water used in the experiments was purified using a 

Barnstead (Dubuque, IA, USA) ROpure LP® reverse osmosis unit with a high-flow 

cellulose acetate membrane (Barnstead D2731) followed by a Barnstead D3804 

NANOpure® organic/colloid removal and ion-exchange packed-bed system. The 

resulting purified water (deionized water) had a specific conductivity of 18 mS/cm. 

Buffer and sample solutions were filtered through 0.2-µm membrane (Sartorius, 

Göttingen, Germany). 

3. 2. Preparation of Cyt c-MIP Nanofibers  

3.2. 1.  Production of Bacterial Cellulose Nanofibers 

The production of BC was performed by growing A. xylinum (ATCC 10245) in 

Hestrin–Schramm medium. The medium containing 20 g/L glucose, 10 g/L 

bactopeptone, 10 g/L yeast extract, 4 mM KH2PO4 and 6 mM K2HPO4 is used for 

the production of BC in static culture. The pH of the medium was adjusted to pH 

5.1–5.2 using 1 M HCl. The inoculum was prepared by growing A. xylinum at 28 

°C for 3 days using a rotary shaker. The BC nanofiber formation was led to occur 

over a period of 7 days after inoculating subculture in the proportion 1:10 in petri 

dishes statically. Then, the synthesized BC nanofibers were washed with 1 M 

NaOH solution at 70 °C for 90 min to remove all the microorganisms inside the 



 33 

membrane. Finally, the BC nanofibers were redispersed in distilled water and 

stored at room temperature [172]. 

3.2. 2. Synthesis of N-methacryloyl L-histidine methylester (MAH) 

The synthesis of MAH was described elsewhere [173] and the experimental 

procedure used was as follows: 5.0 g of L-histidine methylester and 0.2 g of 

hydroquinone were dissolved in 100 mL dichloromethane. This solution was 

cooled to 0 °C, 12.7 g of triethylamine was added to this solution, 5.0 mL of 

methacryloyl chloride was poured into this solution slowly and then it was stirred 

magnetically at room temperature for 2 h. At the end of the chemical reaction, 

extraction with 10% NaOH solution was performed to remove the unreacted 

methacryloyl chloride. The aqueous phase was evaporated and the residue was 

dissolved in ethanol.  

3.2.2. 1. Characterization Studies of MAH 

ATR-FTIR spectrum of MAH monomer was obtained by FTIR spectrometer (FTIR 

8000 series, Shimadzu, Japan).   

3.2. 3. Synthesis of MAH-Cu(II) Metal-chelate Monomer  

In order to prepare MAH-Cu (II) complex, aqueous solutions of MAH (0.01 mmol) 

and copper ions (source: Cu(NO3)2 2.5 H2O) were mixed to prepare this complex 

with two different molar ratios, which was 1:1 and 2:1. Then the mixture was 

allowed to react at 25 °C for 1 h under stirring.  

3.2.3. 1. Characterization Studies of MAH-Cu(II) Complex 

The formation of MAH-Cu(II) complex was determined with UV-vis 

Spectrophotometer (Shimadzu UV-1601, Shimadzu Corp., Kyoto, Japan). The 

samples prepared with two different monomer/metal ion molar ratio (2:1 and 1:1) 

were recorded between 220-600 nm.  

3.2. 4. Preorganization of MAH-Cu(II) Monomer with Cyt c  

For the synthesis of MAH-Cu(II)-Cyt c complex, the buffer system to solubilize 

cytochrome c molecules was selected as pH 7 phosphate buffer since surface 

histidines are largely unprotonated and available to coordinate with the metal ion 

at this pH [174]. After mixing cytochrome c solution (1 mL) with metal chelate 



 34 

monomer, MAH-Cu(II) (10 mL), the newly prepared complex, MAH-Cu(II)-Cyt c 

was left to react at 25 °C for 1 h under gentle stirring. 

3.2. 5. Preparation of Cyt c-MIP Nanofibers 

In order to achieve the creation of MIP layer onto the nanofibers, bacterial 

cellulose nanofiber membranes were reacted with 10% w/w of 3-MPS (3-

methacryloxypropyltrimethoxysilane) in toluene medium at 80 °C for 5 h. Then the 

resultant membranes were washed with methanol [175]. The reacted membranes 

were placed in petri dish to contact with MAH-Cu(II)-Cyt c complex for 30 min. 

After the addition of MBAAm as a cross-linker, this polymerization syrup was 

bubbled with nitrogen gas for 5 min. The initiator, AIBN (0.5 mmol in 100 µL DMF) 

was added under of UV light source (100 W, 365 nm) at room temperature. After 

the polymerization time of 30 min, the MIP layer coated nanofibers were taken out 

of the petri dish and washed with distilled water to remove the unreacted 

chemicals and stored at 4 °C to use at further experiments. The template 

molecules were removed with 1 M NaCl solution for 2 h. The non-imprinted 

nanofibers (NIP) were prepared with the same procedure but the absence of 

template molecules. The formation of recognition sites was presented 

schematically below.  

3.2.5. 1. Characterization Studies of Cyt c-MIP Nanofibers 

FTIR Studies 

The characteristic functional groups of Cyt c imprinted nanofibers were 

characterized by FTIR-attenuated total reflectance (ATR) spectroscopy (FTIR 

8000 Series, Shimadzu, Japan).  

Surface Morphology 

The surface morphology of the imprinted cellulose nanofibers was investigated 

with scanning electron microscope (SEM). The samples were lyophilized before 

being analysed. The samples were then sputtered with a thin layer of gold before 

SEM measurement.  

Contact Angle Measurements 

Contact angle of Cyt c-MIP nanofibers was evaluated with KRUSS DSA 100 

(Hamburg, Germany). It was measured with Sessile Drop method by dropping 1 



 35 

drop of water onto nanofibers. The measurements were the average of the 20 

measurements. 

3. 3. Batch Adsorption Studies 

3.3. 1. Rebinding Experiments 

Adsorption of cytochrome c on the imprinted and non-imprinted nanofibers from 

aqueous solutions was investigated in batch-wise. The nanofibers (16 mg±2.5 mg) 

were incubated with 3 mL of cytochrome c solutions for 1 h at 20 rpm. To observe 

the optimum conditions of polymerization, the effect of monomer/template ratio, 

total monomer ratio and polymerization time with respect to adsorption capacity 

were investigated. The effects of cytochrome c concentration, pH, temperature, 

NaCl concentration on the adsorption capacity was also studied. Cytochrome c 

concentration was changed between 0.075-2.0 mg/mL. The effects of temperature 

were investigated for the temperatures 4, 15, 25 and 37 °C. The effect of ionic 

strength on adsorption capacity was carried out in salt-free solution and then in 

aqueous NaCl solutions with concentration of 0.001-0.1 M. Adsorbed amount of 

cytochrome c was determined by visible spectrophotometry at 409 nm. The 

calibration curve can be seen in Appendix. The protein adsorption capacity was 

calculated from mass balance according to the following equation: 

Q= C0 –C V
m

                                                      (1) 

where Q is the adsorbed amount of Cyt c per unit mass (mg/g), C0 and C are the 

amount of Cyt c before and after adsorption process respectively (mg/mL); V is the 

volume of cytochrome c solution (mL) and m is the dry mass of Cyt c-MIP 

nanofibers used (mg). 

The desorption of the adsorbed Cyt c from the imprinted nanofibers was 

performed in a batch system. The nanofibers were incubated in 1 M NaCl solution 

for 1 h at room temperature under stirring at 20 rpm with rotator.  

3.3. 2. Selectivity Studies 

In order to demonstrate the selectivity of the Cyt c-MIP nanofibers in batch system 

toward template protein Cyt c (pI (isoelectric point)=10.6, MW=12.3 kDa), non-

template proteins bearing surface histidine(s) such as bovine serum albumin 

(BSA, pI= 4.9, MW=67.0 kDa), hemoglobin (Hb, pI=6.7, MW=64.5 kDa), myoglobin 



 36 

(Myo, pI=6.8, MW=17.0 kDa) and lysozyme (Lys, pI=10.5, MW=14.6 kDa) were 

used with initial concentration of 0.2 mg/mL. The concentration of proteins, i.e. 

unadsorbed, was measured by UV-vis spectroscopy at 280 nm for BSA and Lys, 

409 nm for Myo and 406 nm for Hb. All the calibration curves belonging to each 

protein was shown in Appendix. In order to show the contribution of metal ions to 

selectivity properties of MIP nanofibers, the polymerization process without metal 

ion was also performed to obtain M(Cyt c). For the preparation of this MIP 

nanofiber, MAH monomers were complexed with Cyt c directly. 

3. 4. Fabrication of QCM Nanosensors  

The AT cut (5.0 MHz) QCM sensor chip was washed with piranha solution (7:3 

H2SO4 : H2O2, v/v). This solution was treated with gold surface for 20 s and then 

washed with ethanol and water and dried with N2 atmosphere. The prepared Cyt 

c-MIP nanofibers were pulped with the rotation speed of 12000 r/min by a 

homogenizer (T10, Ika Labortechnik, Germany) for 10 min at 25 °C [168]. Then 75 

µL of the treated mixture was dispensed onto the cleaned surface of the electrode 

of the QCM chip with micropipette. The QCM sensor casted with Cyt c-MIP 

nanofiber was dried at 60 °C for 1 h in vacuum. The entire procedure of Cyt c-MIP 

QCM nanosensor was presented schematically below. NIP QCM nanosensor was 

prepared using the same procedure with NIP nanofibers. 

Figure 3. 1.  Schematic presentation of preparation strategy of Cyt c-MIP QCM 

nanosensors. 



 37 

3.4. 1. Surface Characterization of QCM Nanosensors 

In order to characterize the surfaces of QCM nanosensors, atomic force 

microscope (AFM) was used. AFM observations were carried out with AFM 

(Nanomagnetics Instruments, Oxford, UK) in tapping mode. The AFM 

measurements were performed in high resolution such as 4096 x 4096 pixels with 

free cantilever interferometer. The Cyt c-MIP QCM nanosensors and NIP QCM 

nanosensors were placed onto sample holder with double-side carbon strip. All 

observations were taken using tapping mode in air atmosphere. The oscillation 

frequency, vibration amplitude and free vibration amplitude was applied as 341.30 

kHz, 1 VRMS and 2 VRMS. Samples were scanned to obtain the view of the area of 2 

µm x 2 µm using 2 µ/s scanning rate and 256 x 256 pixels resolution. 

3.4. 2. Kinetic Studies with QCM Nanosensors 

To perform the real-time monitoring of QCM nanosensors, different concentrations 

of Cyt c solutions (1-75 µg/mL) were used in the system, which constitute 

frequency counter, oscillator, peristhaltic pump and computer. The experimental 

procedure is as follows: firstly, Cyt c-MIP QCM nanosensors were washed with 

distilled water (50 mL). Then the equilibration was performed using pH 7 

phosphate buffer and the resonance frequency was determined (f0). After 

equilibration process for 3 min, different concentrations of Cyt c solutions (5 mL) 

were given to the system with the flow rate of 1 mL/min. When the resonance 

frequency came to the equilibrium value (approximately 90 min), desorption was 

carried out with 1 M NaCl solution (5 mL, 1 mL/min). The nanosensors were 

washed with distilled water and then with phosphate buffer to equilibrate. The 

resonance frequencies were measured by the QCM digital controller. For each of 

the concentration of Cyt c solution, the adsorption-desorption cycle was utilized. 

The data was analyzed with Software of QCM (Maxtek). 

3.4. 3. Selectivity Studies of QCM Nanosensors 

The non-template proteins that were used in batch rebinding experiments were 

also utilized to show the selectivity of QCM nanosensors. These protein solutions 

were prepared in phosphate buffer (pH:7.0). The resonance frequencies were 

determined for these selectivity experiments. The data was analyzed with 

Software of QCM (Maxtek). 



 38 

4. RESULTS AND DISCUSSION 

The production of biological materials is becoming increasingly attractive and 

developing field. However the isolation and purification steps of the targeted 

molecule often requires expensive treatments which typically account up to 80% of 

the total cost for production [176]. Thus, in order to overcome the high purification 

costs of biological materials there is great demand for the development of 

adsorbents with high specificity and high recognition capacity. Surface imprinting 

is a promising method to create highly specific recognition sites of analyte on the 

surface of the polymer. Nanofibers have been gaining more attention since they 

avoid intraparticle diffusion resistances having high surface area. Surface 

imprinting which is a molecular imprinting process performing onto a solid support 

surface and adopting nanofibers having high surface area are two effective 

approaches to overcome mass transfer resistances especially for 

biomacromolecules like proteins. Fast separation of proteins is very useful for 

many biotechnological areas such as quality control and on-line monitoring [2]. 

The aim of this thesis is to find a cost effective and reusable affinity nanofibers 

having high recognition capacity and high selectivity for purification of Cyt c. 

Surface imprinting has been widely used molecular imprinting method since it 

improves the protein adsorption performance of MIPs by reducing mass transfer 

resistances and enables easy removal of template [5]. In this thesis, surface 

imprinted bacterial cellulose nanofibers for cytochrome c recognition, which was 

abbreviated as Cyt c-MIP, was prepared successfully for efficient purification of 

Cyt c from aqueous solutions.  

4. 1. Preparation of Surface Imprinted MIP Nanofibers 

4.1.1. Synthesis of MAH-Cu(II) Complex 

Metal chelating monomer MAH having imidazole group of chelating property with 

transition metals was complexed with metal ion Cu(II) in molar ratios of 1:1 and 2:1 

and characterized with UV-vis spectrum (Figure 4.1) [177]. It was determined that 

MAH monomer and copper ions were complexed because of the decrement of 

band absorbances [178]. The absorbance of MAH-Cu(II) complex prepared in the 

molar ratio of 1:1 was lower than that of the molar ratio of 2:1. Thus it was 

determined that MAH-Cu(II) complex prepared in the molar ratio of 1:1 namely 



 39 

1MAH-1Cu was selected and it was used for the rest of the study.  

 
Figure 4. 1. UV-vis spectra of MAH and MAH-Cu(II) complexes. 

 

4.1.2. Characterization of Cyt c-MIP nanofibers 

The chemical structure of MAH monomer was characterized by ATR-FTIR. The 

specific bands of MAH monomer were determined as amide bands at 1740 cm−1, 

and 1677 cm−1 (Figure 4.2) [179]. The broad band presenting in the region of 

3600-3000 cm-1 correspond to the O-H streching frequencies of cellulose and the 

band at around 1430 cm-1 is assigned to a symmetric CH2 bending vibration and 

the C-O-C glycosidic ether band arises at ~1105 cm-1 (Figure 4.3) [180]. ATR-FTIR 

spectrum of Cyt c-MIP nanofibers was given below representing characteristic 

amide bands of MAH that occur at 1539 and 1633 cm-1 (Figure 4.4). 

 

 

0 

0.2 

0.4 

0.6 

0.8 

1 

220 320 420 520 

A
bs

or
ba

nc
e 

Wavelength, nm 

MAH 

1MAH-1Cu 

2MAH-1Cu 



 40 

 
Figure 4. 2. ATR-FTIR spectrum of MAH monomer. 

 
 

 
Figure 4. 3. ATR-FTIR spectrum of BC. 

 



 41 

 
Figure 4. 4. ATR-FTIR spectrum of Cyt c-MIP nanofibers. 

 
Surface morphology of Cyt c-MIP nanofibers that were used for batch rebinding 

studies was investigated with SEM (Figure 4.5) indicating that resultant Cyt c-MIP 

bacterial cellulose nanofibers were continuous and randomly oriented as porous 

structure of 3-D non-woven interconnected network nanofibrous mat. As seen from 

the SEM photograph, it was evidently considered that the bacterial cellulose 

nanofibers provide excellent surface area for easy diffusion of cytochrome c 

molecules into and out of the entire membrane facilitating mass transfer properties 

of adsorption process. This nanoweb structure composed of nanofibers 

contributes to the rapid diffusion of template molecules into the recognitive MIP 

layer enhancing rebinding kinetics [181]. Also it was clear that there was no 

bacteria and debris in the polymer structure which indicating that washing 

procedure used was successful to remove all the microorganisms.  

The contact angle measurements were performed to demonstrate the hydrophilic 

character of MIP nanofibers (Figure 4.6). The average contact angle of Cyt c-MIP 

nanofiber was found as 49.72°. 

 



 42 

 
Figure 4. 5. SEM image of Cyt c-MIP nanofibers. 

 

                        
Figure 4. 6. Contact angle measurment of Cyt c-MIP nanofiber. 

 

4. 2.  Study of Conditions of Polymerization 

In order to evaluate the Cyt c adsorption capacity of MIP nanofibers, batch binding 

experiments were performed in a well-mixed vessel. The importance of polymer 

composition for molecular imprinting was reviewed elsewhere [182]. The careful 

choice of functional monomer is very important to obtain highly spesific recognition 



 43 

binding sites. Actually the selection of functional monomer is determined by the 

structure of the template. The optimization of monomer/template ratio is crucial to 

create binding sites with high fidelity. For non covalent imprinting process it should 

be achieved empirically. For instance, if there is only one interaction point between 

template and monomer, the amount of template gets saturation after all the 

functional monomer is complexed with template molecules. However there is no 

saturation when there is two point interaction between template and monomer. 

Functional monomer/crosslinker ratio is also a critical parameter since high 

percentage of functional monomer will cause non spesific adsorption and also low 

amount of cross linker will cause loose polymer structure resulting insufficient 

imprinting and low percentage of functional monomer will cause weak interactions 

with template giving low capacity of binding [29], [183], [20].  

4.2. 1. Effect of Monomer/Template Ratio 

It is significant to determine the amount of metal chelating monomer, for this 

project MAH-Cu(II), templating Cyt c to form a cav