Molecularly imprinting strategies for

hydrophilic compounds and

macromolecules

PhD Student: Laura – Elena Gliga

Thesis Supervisor: Prof. Dr. Radu
Opreanth
July 24 , 2023, Cluj-Napoca

“Iuliu Haţieganu” University of Medicine and Pharmacy

Cluj-Napoca, Romania
Department of Analytical Chemistry, Faculty of Pharmacy
 Presentation Structure
1. Important biological macromolecules
State of the 2. Cell-free nucleic acids detection and quantification techniques
art 3. Specific analytical methods for affinity studies in the field of nucleic
acids, employed in the present work

1. Improved enantioselectivity for atenolol employing pivot based

molecular imprinting
2. Electrochemical platform for the detection of adenosine using a
Personal
sandwich-structured molecularly imprinted polymer-based sensor
contributions
3. Analytical perspectives in the study of polyvalent interactions of free
and surface-bound oligonucleotides and their implications in affinity
2
biosensing
 Presentation Structure
1. Important biological macromolecules
State of the 2. Cell-free nucleic acids detection and quantification techniques
art 3. Specific analytical methods for affinity studies in the field of nucleic
acids, employed in the present work

1. Improved enantioselectivity for atenolol employing pivot based

molecular imprinting
2. Electrochemical platform for the detection of adenosine using a
Personal
sandwich-structured molecularly imprinted polymer-based sensor
contributions
3. Analytical perspectives in the study of polyvalent interactions of free
and surface-bound oligonucleotides and their implications in affinity
3
biosensing
 State of the art
1. Important biological macromolecules
Biomacromolecules: Lipid Carbohydrate Protein

- naturally occurring polar compounds of large size and high molecular

weight
- complex structure – biopolymers (except lipids)
Nucleic acid
- vital role in almost all biophysical processes of living organisms

Nucleic acids (Polynucleotides): Nucleotide

- store and transfer genetic information and guide protein synthesis Triphosphate

- long polymer structure → nucleotide

- Deoxyribonucleic acid (DNA) Nitrogen base
- purine
- two-stranded helical structure – H bonds between nitrogen bases - pyrimidine
Sugar
- Ribonucleic acid (RNA) - β-D-ribose (RNA)
- β-2-deoxy-D-ribose (DNA)
- single stranded nucleic acid
- higher chemical lability compared to DNA 4
 State of the art
1. Important biological macromolecules
Cell-free nucleic acids:
- high stability in body fluids → valuable as biomarkers for different
pathological processes (tumors, inflammatory diseases, stroke, sepsis and
tissue trauma)
Cell-free
- found in blood, urine, saliva, breastmilk, pleural effusion, amniotic fluid, etc.) fetal
DNA
• DNA species: damaged DNA fragments – cell-free nuclear DNA, cell-free
mitochondrial DNA, circulating tumor DNA, cell-free fetal DNA, etc.
• RNA species: coding messenger RNA, microRNA, ribosomal RNA, etc.

Nucleic acid constituents – nucleotides, nucleosides, purine and

pyrimidine bases
- associated with metabolic regulation, synthesis of numerous biomolecules and
other vital processes in cell physiology
5

https://www.prenatalscreeningontario.ca
 Presentation Structure
1. Important biological macromolecules
State of the 2. Cell-free nucleic acids detection and quantification techniques
art 3. Specific analytical methods for affinity studies in the field of nucleic
acids, employed in the present work

1. Improved enantioselectivity for atenolol employing pivot based

molecular imprinting
2. Electrochemical platform for the detection of adenosine using a
Personal
sandwich-structured molecularly imprinted polymer-based sensor
contributions
3. Analytical perspectives in the study of polyvalent interactions of free
and surface-bound oligonucleotides and their implications in affinity
6
biosensing
 State of the art
2. Cell-free nucleic acids detection and quantification
Standard detection methods for nucleic acids:
techniques

Cell-free DNA: Cell-free RNA:

- Quantitative Polymerase Chain Reaction (qPCR) – “gold - Reverse Transcription qPCR (RT-qPCR) – “gold
standard” method standard” method for diagnostic purposes
- Next-generation DNA sequencing (NGS) - RNA-Sequencing (RNA-Seq)
- Cancer personalized profiling by deep sequencing (CAPP-
Seq)

- Liquid Chromatography → Nucleic acids constituents (nucleotides, nucleosides, etc.)

Foster holistically
Rapid and accurate biomarker identification is critical for diagnosis, effective management and medical treatment of numerous
superior
severe diseases methodologies

7
Sensors → Electrochemical
sensors
 State of the art
2. Cell-free nucleic acids detection and quantification
Electrochemical
techniquessensors for nucleic acids analysis:
- Electroactive surface
- Electroanalytical method
- Molecular recognition element
• defines the specific interaction between two or more molecules (by non-covalent bonding)
• have a major influence on the sensor’s specificity, stability, reusability, sensitivity, and cost of production

Synthetic biorecognition Naturally occurring

elements biorecognition elements
Aptamer
• comparable selectivity Molecularly Nucleic acid • high affinity and specificity
• higher stability imprinted Foster holistically
• tailor-made for a wide polymers Antibody •
superior stability issues, complicated
range of analytes (MIPs) methodologies and costly production
• cost-effective Enzyme processes

8
 State of the art
2. Cell-free nucleic acids detection and quantification
techniques
Molecularly imprinted polymers (MIPs) - polymer composites with predetermined selectivity and high affinity

Advantages: Imprinting Applications: Macromolecules

- high affinity and mechanisms: - SPE cartridges imprinting challenges:
selectivity for a wide - covalent - LC/CE stationary phase - lack of available water-
range of analytes - non-covalent - chiral analysis soluble monomers
- excellent stability - semi-covalent - drug delivery - significant non-specific
- low-cost - metal ion-mediated - Recognition elements binding
– sensors - high instability

New imprinting strategies for polar compounds and macromolecules:

• Metal ion-mediated molecular imprinting (MMMI) Foster holistically
superior
• Synthesis of MIP as nanoparticles (nanoMIPs) methodologies

• Double recognition MIPs – MIP/Aptamer → high degree of binding site homogeneity

9

Akgönüllü S, Kılıç S, Esen C, Denizli A. Molecularly Imprinted Polymer-Based Sensors for Protein Detection. Polymers 2023; 15:629.
 Presentation Structure
1. Important biological macromolecules
State of the 2. Cell-free nucleic acids detection and quantification techniques
art 3. Specific analytical methods for affinity studies in the field of nucleic
acids, employed in the present work

1. Improved enantioselectivity for atenolol employing pivot based

molecular imprinting
2. Electrochemical platform for the detection of adenosine using a
Personal
sandwich-structured molecularly imprinted polymer-based sensor
contributions
3. Analytical perspectives in the study of polyvalent interactions of free
and surface-bound oligonucleotides and their implications in affinity
10
biosensing
 State of the art
3. Specific analytical methods for affinity studies in the field of nucleic acids, employed in the
- Binding affinity →
present the specificity of the biorecognition element
work
- A robust and selective affinity is required → the thermodynamic and kinetic properties of the interaction are of great value when
selecting the appropriate recognition component.

Analytical methods for affinity studies:

Separation techniques
High performance affinity chromatography (HPAC)
Affinity capillary electrophoresis (ACE)
Equilibrium dialysis (ED), etc.

Foster holistically
Other techniques superior
methodologies
Spectroscopic approaches (UV, NMR, etc.)
Surface plasmon resonance (SPR)
11
Isothermal titration calorimetry (ITC), etc.
 Objectives
Innovative molecularly imprinting strategies for high polarity
compounds
1. Improved enantioselectivity for atenolol employing pivot based
molecular imprinting
2. Electrochemical platform for the detection of adenosine using a
Nov 20XX May 20XX
sandwich-structured molecularly imprinted polymer-based sensor
Disseminate Deploy strategy
4 standardized metrics networks with
compelling e-

Experimental
business needs

studies
Sep 20XX Thermodynamic and kinetic properties of the interaction between
Synergize scalable ssDNA sequences, acting as ligands and targets in the development
e-commerce Jan 20XX Mar 20XX
of biosensors
Coordinate e- Foster holistically
3. business
Analytical perspectives in the study
applications of polyvalent interactions of free
superior
methodologies
and surface-bound oligonucleotides and their implications in affinity
biosensing
12
4. Hybridization thermodynamics of DNA oligonucleotide cognates
 Presentation Structure
1. Important biological macromolecules
State of the 2. Cell-free nucleic acids detection and quantification techniques
art 3. Specific analytical methods for affinity studies in the field of nucleic
acids, employed in the present work

1. Improved enantioselectivity for atenolol employing pivot based

molecular imprinting
2. Electrochemical platform for the detection of adenosine using a
Personal
sandwich-structured molecularly imprinted polymer-based sensor
contributions
3. Analytical perspectives in the study of polyvalent interactions of free
and surface-bound oligonucleotides and their implications in affinity
13
biosensing
 Personal contributions

Objective:
• Investigation of the favourable kosmotropic effect of a
ternary complex:
polar chiral template (eutomer of atenolol)
Improved functional monomer
central metal ion → well-defined, spatially directional
enantioselectivity for coordinate bonds.

atenolol employing
Generalities:
pivot based molecular • Atenolol → polar β-blocker
imprinting - poor enantioselectivity reported by various chiral selectors
- good chelating agent
 Personal contributions
1. Improved enantioselectivity for atenolol employing pivot based molecular imprinting

Working protocol:
- MIP and NIP synthesis and evaluation

Imprinting Polymerization Polymer

Polymer type
strategies components: evaluation:

- non-covalent - bulk (photo- - Metal ion: Co (II), Ni - by HPLC

imprinting polymerization/thermal (II), Cu (II), etc. - various solvent
- metal ion-mediated polymerization → - Functional monomer: systems were tested as
imprinting slurry-packed into HPLC MAA, 4-Vpy, etc. mobile phase: ACN, ACN
columns) - Cross-linker: EDMA, + aqueous buffers
- monolith (in situ etc.
thermopolymerization, - Porogenic solvent: - α, separation factor,
in HPLC columns) DMF/DMSO, ACN was determined

15

Bodoki A, Iacob B-C, Gliga L-E, Oprean L, Spivak D, Gariano N, et al. Improved Enantioselectivity for Atenolol Employing Pivot Based Molecular Imprinting. Molecules 2018;23:1875.
 Personal contributions
1. Improved enantioselectivity for atenolol employing pivot based molecular imprinting

Results:

Ternary metal complexes of ATNL

UV-Vis spectroscopy → simple, fast, cost-effective and relatively straightforward instrumental method for assessing the formation of
the ternary complex

Co(II) – 4-Vpy
Cu(II) – MAA.

16

Bodoki A, Iacob B-C, Gliga L-E, Oprean L, Spivak D, Gariano N, et al. Improved Enantioselectivity for Atenolol Employing Pivot Based Molecular Imprinting. Molecules 2018;23:1875.
 Personal contributions
1. Improved enantioselectivity for atenolol employing pivot based molecular imprinting

Results:

Preparation of MIPs
• Non-covalent molecular imprinting

Imprinting Form of the Polymerization

mechanisms polymer components
non-covalent - bulk - Functional monomer:
imprinting MAA, AM, VIM, 4-VPy
- monolith - low enantioselectivity (α = 1.0–1.32)
⇩ - Additives: cross-linkers, ILs
DMF/ α=
based on the ability of - Porogenic solvent: M6 S-ATNL 4-VPy EDMA IL
DMSO 1.30
ATNL to interact with H-
bonding monomers - ! aprotic: ACN, DMF, α=
DMSO M18 S-ATNL MAA EDMA ACN IL
1.32
- protic: MeOH, butanol
17

Bodoki A, Iacob B-C, Gliga L-E, Oprean L, Spivak D, Gariano N, et al. Improved Enantioselectivity for Atenolol Employing Pivot Based Molecular Imprinting. Molecules 2018;23:1875.
 Personal contributions
1. Improved enantioselectivity for atenolol employing pivot based molecular imprinting

Results:

Preparation of MIPs
• Metal ion-mediated molecular imprinting (MMMI) - monomers
are positioned around the template via coordinate bonds
restraining the free motion of the species

Imprinting Form of the Polymerization

mechanisms polymer components
Metal ion-mediated - bulk - Co(II), Cu(II), Ni(II) Enantioselectivity (α) and chromatographic retention (k’s/k’r of
molecular imprinting MMMI)
(MMMI) - monolith - Functional monomer:
MAA, AM, VIM, 4-VPy
S- 4- DMF/ α=
M2 Co(II) EDMA IL
- Additives: cross-linkers ATNL VPy DMSO 1.60
(NOBE, TRIM, EDMA), ILs
- Porogenic solvent: Best performing
- aprotic: DMF, DMSO MIP monolith
18

Bodoki A, Iacob B-C, Gliga L-E, Oprean L, Spivak D, Gariano N, et al. Improved Enantioselectivity for Atenolol Employing Pivot Based Molecular Imprinting. Molecules 2018;23:1875.
 Presentation Structure
1. Important biological macromolecules
State of the 2. Cell-free nucleic acids detection and quantification techniques
art 3. Specific analytical methods for affinity studies in the field of nucleic
acids, employed in the present work

1. Improved enantioselectivity for atenolol employing pivot based

molecular imprinting
2. Electrochemical platform for the detection of adenosine using a
Personal
sandwich-structured molecularly imprinted polymer-based sensor
contributions
3. Analytical perspectives in the study of polyvalent interactions of free
and surface-bound oligonucleotides and their implications in affinity
19
biosensing
 Personal contributions

Objective:
• An innovative sandwich-type MIP is proposed → 2 polymeric
layers: covalent + non-covalent interactions
Electrochemical platform
• Development of a MIP-based biomimetic electrochemical sensor
for the detection of capable of detecting, with high specificity, low-levels of urinary
adenosine
adenosine using a
sandwich-structured
Generalities:
molecularly imprinted
• Adenosine (6-amino-9-β-D-ribofuranosyl-9-H-purine)
polymer-based sensor - endogenous purine nucleoside involved in multiple biochemical
processes
- circulating biomarker in different types of cancer
 Personal contributions
2. Electrochemical platform for the detection of adenosine using a sandwich-structured
molecularly imprinted polymer-based sensor

Working protocol:

21
Gliga L-E, Iacob B-C, Cheșcheș B, Florea A, Barbu-Tudoran L, Bodoki E, et al. Electrochemical platform for the detection of adenosine using a sandwich-structured molecularly imprinted polymer-based sensor. Electrochim
Acta 2020;354:136656.
 Personal contributions
2. Electrochemical platform for the detection of adenosine using a sandwich-structured
molecularly imprinted polymer-based sensor

Results:
MIP film characterization – SEM characterization

After the electrodeposition of 1st NIP MIP before template extraction MIP after template extraction
polymeric layer

22
Gliga L-E, Iacob B-C, Cheșcheș B, Florea A, Barbu-Tudoran L, Bodoki E, et al. Electrochemical platform for the detection of adenosine using a sandwich-structured molecularly imprinted polymer-based sensor. Electrochim
Acta 2020;354:136656.
 Personal contributions
2. Electrochemical platform for the detection of adenosine using a sandwich-structured
molecularly imprinted polymer-based sensor

Results:
MIP film characterization – Impedimetric characterization
A B

bare electrode after 1st layer after ADO being anchored after 2nd layer after template removal after template rebinding

(A) Nyquist plots, the corresponding equivalent circuit in the inset, (B) – zoomed Nyquist plot

23
Gliga L-E, Iacob B-C, Cheșcheș B, Florea A, Barbu-Tudoran L, Bodoki E, et al. Electrochemical platform for the detection of adenosine using a sandwich-structured molecularly imprinted polymer-based sensor. Electrochim
Acta 2020;354:136656.
 Personal contributions
2. Electrochemical platform for the detection of adenosine using a sandwich-structured
molecularly imprinted polymer-based sensor

Results:
MIP film characterization – Voltammetric characterization

a
a
b
d
a c
b b
c

c
d

Cyclic voltammograms of (a) bare GCE, (c) Cyclic voltammograms (a) after MIP template DPV (a) after MIP template removal, (d)
after 1st and (b) 2nd polymeric layer removal, (d) after NIP exposed to acidified after NIP exposed to acidified MeOH, (b)
electrodeposition MeOH, (b) after MIP ADO rebinding, (c) after after MIP ADO rebinding, (c) after NIP ADO
NIP ADO rebinding rebinding 24
Gliga L-E, Iacob B-C, Cheșcheș B, Florea A, Barbu-Tudoran L, Bodoki E, et al. Electrochemical platform for the detection of adenosine using a sandwich-structured molecularly imprinted polymer-based sensor. Electrochim
Acta 2020;354:136656.
 Personal contributions
2. Electrochemical platform for the detection of adenosine using a sandwich-structured
molecularly imprinted polymer-based sensor

Results:
Sensor optimization – 1st polymeric film composition (boronic acid type and concentration)

∆I (µA)* Std. dev

3-TBA (10 mM) 14.00 0.81

3-APBA (2.5 mM) 11.69 1.22

BPBAresponse
Sensor (2.5 mM)(∆I) using the5.21 0.71for each
optimum concentration
boronic acid derivative

Influence of boronic acid functional MIP selectivity towards ADO and

monomer:BTh molar ratio on the interferents (ADE, GUO) when the
recorded electrochemical signal functional monomer is black – 3-TBA
10 mM or red shaded – 3-APBA 2.5 mM

3-TBA 3-APBA BPBA 2.5 mM 3-APBA

25
Gliga L-E, Iacob B-C, Cheșcheș B, Florea A, Barbu-Tudoran L, Bodoki E, et al. Electrochemical platform for the detection of adenosine using a sandwich-structured molecularly imprinted polymer-based sensor. Electrochim
Acta 2020;354:136656.
 Personal contributions
2. Electrochemical platform for the detection of adenosine using a sandwich-structured
molecularly imprinted polymer-based sensor

Results:
Sensor optimization – 2nd polymeric film composition (I3AA:ADO ratio)

I3AA:ADO = 1:1

The influence of the I3AA:ADO ratio on the recorded redox signal

26
Gliga L-E, Iacob B-C, Cheșcheș B, Florea A, Barbu-Tudoran L, Bodoki E, et al. Electrochemical platform for the detection of adenosine using a sandwich-structured molecularly imprinted polymer-based sensor. Electrochim
Acta 2020;354:136656.
 Personal contributions
2. Electrochemical platform for the detection of adenosine using a sandwich-structured
molecularly imprinted polymer-based sensor

Results:
Sensor optimization – Template removal time/Template rebinding time

Optimum extraction time: 10 min in

Optimum rebinding time: 20 min
MeOH: Acetic Acid 100% = 90:10
27
Gliga L-E, Iacob B-C, Cheșcheș B, Florea A, Barbu-Tudoran L, Bodoki E, et al. Electrochemical platform for the detection of adenosine using a sandwich-structured molecularly imprinted polymer-based sensor. Electrochim
Acta 2020;354:136656.
 Personal contributions
2. Electrochemical platform for the detection of adenosine using a sandwich-structured
molecularly imprinted polymer-based sensor

Results:
Analytical performance – Sensor calibration
Linear dependence of the sensor’s
response (a) MIP, (b) NIP with ADO
∆I = 966151CM (± 34190.52) + 6.21 (± 0.06) concentration
R2=0.9978

• Linear range: 0.37 μM – 37.4 µM

• LOQ: 0.64 µM
• LOD: 0.21 µM

28
Gliga L-E, Iacob B-C, Cheșcheș B, Florea A, Barbu-Tudoran L, Bodoki E, et al. Electrochemical platform for the detection of adenosine using a sandwich-structured molecularly imprinted polymer-based sensor. Electrochim
Acta 2020;354:136656.
 Personal contributions
2. Electrochemical platform for the detection of adenosine using a sandwich-structured
molecularly imprinted polymer-based sensor

Results:
Analytical performance – Sensor selectivity
Selectivity of the MIP-based sensor and NIP-based sensor towards
MIP
structurally related compounds and biomolecules
NIP

ADO GUO ADE THM INO GLU

CYT URA URD ATP UA

29
Gliga L-E, Iacob B-C, Cheșcheș B, Florea A, Barbu-Tudoran L, Bodoki E, et al. Electrochemical platform for the detection of adenosine using a sandwich-structured molecularly imprinted polymer-based sensor. Electrochim
Acta 2020;354:136656.
 Personal contributions
2. Electrochemical platform for the detection of adenosine using a sandwich-structured
molecularly imprinted polymer-based sensor

Results:
Analytical performance – Sensor reusability
The MIP-based sensor could be reliably used up to 4
repeated ADO analyses , without leading to a significant
decrease of the response signal (<15 %).

30
Gliga L-E, Iacob B-C, Cheșcheș B, Florea A, Barbu-Tudoran L, Bodoki E, et al. Electrochemical platform for the detection of adenosine using a sandwich-structured molecularly imprinted polymer-based sensor. Electrochim
Acta 2020;354:136656.
 Personal contributions
2. Electrochemical platform for the detection of adenosine using a sandwich-structured
molecularly imprinted polymer-based sensor

Results:
Analytical performance – Real sample analysis

Sample Added (µM) Found (µM) Recovery (%)

Adenosine urinary levels in physiological conditions:
<4.5 µM (1.2 µg⋅ml-1) Urine 18.7 19.25 102.98 (±6.12)

Adenosine urinary levels in pathological conditions: 37.4 39.77 106.34 (±7.40)

up to 10 µM – 50 µM

31
Gliga L-E, Iacob B-C, Cheșcheș B, Florea A, Barbu-Tudoran L, Bodoki E, et al. Electrochemical platform for the detection of adenosine using a sandwich-structured molecularly imprinted polymer-based sensor. Electrochim
Acta 2020;354:136656.
 General conclusions – 1st and 2nd
study
• A performant metal ion-mediated molecularly imprinted polymer, in terms of
enantioselectivity for (R)-atenolol, was developed.
1 • The influence of different experimental variables on the chromatographic retention and
enantioselectivity were studied → rigorous selection of polymerization components is vital.

• A new electrochemical sensor was developed for the sensitive and label-free detection of ADO in
biological fluids.
2
• The sensor relies on a sandwich-type MIP film as the recognition element.

Two less explored imprinting strategies for high polarity compounds are proposed.

32
 Presentation Structure
1. Important biological macromolecules
State of the 2. Cell-free nucleic acids detection and quantification techniques
art 3. Specific analytical methods for affinity studies in the field of nucleic
acids, employed in the present work

1. Improved enantioselectivity for atenolol employing pivot based

molecular imprinting
2. Electrochemical platform for the detection of adenosine using a
Personal
sandwich-structured molecularly imprinted polymer-based sensor
contributions
3. Analytical perspectives in the study of polyvalent interactions of free
and surface-bound oligonucleotides and their implications in affinity
33
biosensing
 Personal contributions

3. Analytical perspectives in the Objectives of 3rd and 4th Study:

• Thermodynamic and kinetic investigation:
study of polyvalent interactions of • Binary ssDNA interactions
• Ternary ssDNA interactions
free and surface-bound • ssDNA sequences bound to metallic nanostructures
(GNRs)
oligonucleotides and their • Interactions between ssDNA sequences of various lengths.
implications in affinity biosensing
4. Hybridization thermodynamics of Generalities:
• Nucleic acids (NAs) → recognition elements for sensors
DNA oligonucleotide cognates
 Personal contributions
3. Analytical perspectives in the study of polyvalent interactions of free and surface-bound
oligonucleotides and their implications in affinity biosensing
Working protocol:
MOLECULAR RECOGNITION
ssDNA BINDING STRATEGY
ELEMENTS
Recognition
FOR BIOSENSING APPLICATIONS
sequences
Free oligonucleotides
Target specific
oligonucleotides
A1 A2

Target
sequence CGE
A1 – miR21 A2 – miR21
A1 – miR21 –
A2 ternary
ITC
binary complex binary complex
complex

miR21
Surface-bound oligonucleotides
miR21 –
A2@GNRs A1 – miR21 –
Signal transduction A2@GNRs ternary
A2@GN binary
platform – noble metal complex complex
Rs
surfaces

35
Gliga L-E, Iacob B-C, Moldovean S-N, Spivak DA, Bodoki AE, Bodoki E, et al. Analytical Perspectives in the Study of Polyvalent Interactions of Free and Surface-Bound Oligonucleotides and Their Implications in Affinity
Biosensing. Int J Mol Sci 2023;24
 Personal contributions
3. Analytical perspectives in the study of polyvalent interactions of free and surface-bound
oligonucleotides and their implications in affinity biosensing

Results:
CGE analysis – Binary complex assessment (miR21 – A1)

excess miR21 miR21 – A1

A1 binary
Orange G complex

- A1 conc. – constant (2.22 µM)

- miR21 conc. – progressively increased (0–8.88 µM)
A1 –
miR21
binary
complex

36
Gliga L-E, Iacob B-C, Moldovean S-N, Spivak DA, Bodoki AE, Bodoki E, et al. Analytical Perspectives in the Study of Polyvalent Interactions of Free and Surface-Bound Oligonucleotides and Their Implications in Affinity
Biosensing. Int J Mol Sci 2023;24
 Personal contributions
3. Analytical perspectives in the study of polyvalent interactions of free and surface-bound
oligonucleotides and their implications in affinity biosensing

Results:
CGE analysis – Binary complex assessment – Binding stoichiometry (miR21 – A1, miR21 – A2)

37
Gliga L-E, Iacob B-C, Moldovean S-N, Spivak DA, Bodoki AE, Bodoki E, et al. Analytical Perspectives in the Study of Polyvalent Interactions of Free and Surface-Bound Oligonucleotides and Their Implications in Affinity
Biosensing. Int J Mol Sci 2023;24
 Personal contributions
3. Analytical perspectives in the study of polyvalent interactions of free and surface-bound
oligonucleotides and their implications in affinity biosensing

Results:
CGE analysis – Ternary complex assessment (A1 – miR21 – A2)

Ternary complex – resulting

between 3 free ssDNA sequences
– can be assessed by CGE

A1 – miR21 –
A2 ternary
complex
38
Gliga L-E, Iacob B-C, Moldovean S-N, Spivak DA, Bodoki AE, Bodoki E, et al. Analytical Perspectives in the Study of Polyvalent Interactions of Free and Surface-Bound Oligonucleotides and Their Implications in Affinity
Biosensing. Int J Mol Sci 2023;24
 Personal contributions
3. Analytical perspectives in the study of polyvalent interactions of free and surface-bound
oligonucleotides and their implications in affinity biosensing

Results:
CGE analysis – Binary complex assessment (miR21 – A2@GNRs)

miR21 – A2@GNRs interaction

can’t be detected by CGE

miR21 –
A2@GNRs binary
complex 39
Gliga L-E, Iacob B-C, Moldovean S-N, Spivak DA, Bodoki AE, Bodoki E, et al. Analytical Perspectives in the Study of Polyvalent Interactions of Free and Surface-Bound Oligonucleotides and Their Implications in Affinity
Biosensing. Int J Mol Sci 2023;24
 Personal contributions
3. Analytical perspectives in the study of polyvalent interactions of free and surface-bound
oligonucleotides and their implications in affinity biosensing

Results:
CGE analysis – Ternary complex assessment (A1 – miR21 – A2, A1 – miR21 – A2@GNRs)

A1 – miR21 – A1 – miR21
NOT binary complex
A2@GNRs ternary
detected
complex

40
Gliga L-E, Iacob B-C, Moldovean S-N, Spivak DA, Bodoki AE, Bodoki E, et al. Analytical Perspectives in the Study of Polyvalent Interactions of Free and Surface-Bound Oligonucleotides and Their Implications in Affinity
Biosensing. Int J Mol Sci 2023;24
 Personal contributions
3. Analytical perspectives in the study of polyvalent interactions of free and surface-bound
oligonucleotides and their implications in affinity biosensing

Results:
CGE analysis – Binding Selectivity

NO interaction between miR21 and

low complementarity/random
sequences

41
Gliga L-E, Iacob B-C, Moldovean S-N, Spivak DA, Bodoki AE, Bodoki E, et al. Analytical Perspectives in the Study of Polyvalent Interactions of Free and Surface-Bound Oligonucleotides and Their Implications in Affinity
Biosensing. Int J Mol Sci 2023;24
 Personal contributions
3. Analytical perspectives in the study of polyvalent interactions of free and surface-bound
oligonucleotides and their implications in affinity biosensing

Results:
Microcalorimetric study – Binary interactions (miR21 – A1, miR21 – A2)

42
Gliga L-E, Iacob B-C, Moldovean S-N, Spivak DA, Bodoki AE, Bodoki E, et al. Analytical Perspectives in the Study of Polyvalent Interactions of Free and Surface-Bound Oligonucleotides and Their Implications in Affinity
Biosensing. Int J Mol Sci 2023;24
 Personal contributions
3. Analytical perspectives in the study of polyvalent interactions of free and surface-bound
oligonucleotides and their implications in affinity biosensing

Results:
Microcalorimetric study – Binary interactions with A2@GNRs
A1 10 µM A1 30 µM A1 10 µM + A2 10 µM A2 10 µM (A1 10 µM + A2
+ + random/ + + 10 µM)
miR21 miR21-sp anti-miR21 miR21 miR21 + miR21
30 µM 90 µM 30 µM 30 µM 30 µM * 30 µM
KD
0.38 9.40 1.25 1.55 0.55
(μM) NB
(38.25) (24.56) (11.13) (12.35) (4.84)
(RSD%)
N 1.118 0.973 0.96 1.018 0.45
-
(RSD%) (14.10) (6.09) (10.42) (6.90) (5.08)
ΔG
−36.767 −28.69 −33.7 −33.17 −35.70
(kJ/mol) -
(−2.93) (-2.57) (−0.79) (−0.91) (−0.33)
(RSD%)
ΔH
miR21 + A2@GNRs −201.23 −18.36 −164.93 −174.9 −508.5
(kJ/mol) -
(−4.15) (26.01) (−11.39) (−4.86) (−1.45)
miR21-sp + A2@GNRs (RSD%)
−TΔS
random + A2@GNRs 164.467 −10.33 131.23 141.76 472.76
(kJ/mol) -
(5.72) (30.79) (14.12) (6.01) (1.55)
(RSD%)

43
Gliga L-E, Iacob B-C, Moldovean S-N, Spivak DA, Bodoki AE, Bodoki E, et al. Analytical Perspectives in the Study of Polyvalent Interactions of Free and Surface-Bound Oligonucleotides and Their Implications in Affinity
Biosensing. Int J Mol Sci 2023;24
 Personal contributions
4. Hybridization thermodynamics of DNA oligonucleotide cognates
• ssDNA – MIP-sensors – several strategies (incorporation
Working protocol: of full-length sequences, shorter single sequences or
multiple split fragments) → lack of information regarding
the optimal strategy

Isothermal Titration Calorimetry

Capillary Gel Electrophoresis

44
 Personal contributions
3. Analytical perspectives in the study of polyvalent interactions of free and surface-bound
oligonucleotides and their implications in affinity biosensing

Results:
Binary interactions – Isothermal Titration Calorimetry (ITC)

- A1_23%, A2_23% – 5 nucleotides → NO

interaction with miR21 – 22 nucleotides

- miR21 – A1_77% vs. miR21 – A1_A2 → no

statistically significant difference in binding
affinity

- higher GC content → higher binding affinity

45

- CGE analysis confirmed these interactions

 Personal contributions
3. Analytical perspectives in the study of polyvalent interactions of free and surface-bound
oligonucleotides and their implications in affinity biosensing

Results:
Ternary interactions, Competitive binding studies – Isothermal Titration Calorimetry (ITC)

miR21 – A1_A2 vs. miR21 – A1 + A2 → higher binding affinity, 46

similar thermodynamic signatures
 Personal contributions
3. Analytical perspectives in the study of polyvalent interactions of free and surface-bound
oligonucleotides and their implications in affinity biosensing

Results:
Ternary interactions, Competitive binding studies – Isothermal Titration Calorimetry (ITC)

No need for simultaneous interaction with 2 ssDNAs for ternary 47

complex assessment.
 Personal contributions
3. Analytical perspectives in the study of polyvalent interactions of free and surface-bound
oligonucleotides and their implications in affinity biosensing

Results:
Ternary interactions, Competitive binding studies – Isothermal Titration Calorimetry (ITC)

If the foremost part of miR21 is already hybridized, the unhybridized tail consisting of 5 bases → not sufficient 48
for interaction with other ssDNA sequences, regardless of their length
 General conclusions – 3rd and 4th
study

3 • The complementary electrophoretic and microcalorimetric data provide a quick and

comprehensive representation of the specific or non-specific interactions that occur between
various free and metal surface-bound oligonucleotides in dynamic equilibria.

• Capillary gel electrophoresis resulted as being unappropriated to study the interaction between
metal surface-bound oligonucleotides.

4 • Higher order interactions (ternary) between ssDNA sequences proved to be auspicious →

rigorous selection of the probe NA sequences is mandatory.

Valuable information regarding the interaction between ssDNA sequences is provided.

49
Thank you for
your attention!