Extended Standard Hough Transform for Analytical Line Recognition
Molecular Recognition Using Uncrosslinked Polymers of N-isopropylacrylamide
1
Molecular Recognition Using Uncrosslinked Polymers of N- isopropylacrylamide Alex Papantones N N N N N N Quinoxaline Quinazoline 1,5-naphthyridine λ abs = 310.1 nm λ abs = 314.9 nm λ abs = 306.5 nm Background The molecules involved bind as “host” and “guest” molecules. The two molecules are complementary, and can form a coordination complex between each other that the host molecule cannot form with any other molecule. A good analogy is a keyhole and a key. The guest will easily bond to the host but any other guest molecule will not be able to bind to the host molecule at all. Molecular Recognition Polymerization details Polymers purified by dialysis - For this project concentrations of MAA were 0, 10, or 20 mol% - Reaction reaches completion in minutes - Free Radical synthesis, can use water or acetonitrile as solvent Properties of polyNIPA PolyNIPA has a Lower Critical Solution Temperature (LCST) of 32°C. It is soluble in water below this temperature and will aggregate out of solution at higher temperatures. This process is reversible 4 . Vials of 40 mg/mL polyNIPA 20% MAA in pH 7 HBES buffer at 65°C (left) and 20°C (right) When the polymerization is carried out at high temperature in the presence of a template molecule, the molecules are surrounded by aggregated polymer imprinting that molecule into the polymer structure. Allowing the polymer to relax to the random coil state releases the template molecules during dialysis 5 . Zeta Potential is the electrical potential between the dispersion medium and the stationary area of fluid attached to a dispersed particle. It can also be described as electrical potential in the interfacial double layer (DL) at the location of the slipping plane versus a point in the bulk fluid away from the interface. A value of 30 mV (+ or -) would be considered to be a highly charged, stable 2 -There is a charge on the outer surfaces of polymer interacting with the solution -High zeta potential can tell us precisely at what temp LCST occurs Can often give us clues as to the structure of a polymer aggregate -Measured by Zetasizer Nano instrument using Laser Doppler Velicometry 3 25 30 35 40 45 50 55 60 -35 -30 -25 -20 -15 -10 -5 0 poly(NIPA 80:20 MAA) Temperature (*C) Zeta potential (mV) 15 20 25 30 35 40 45 50 55 60 65 -50 -45 -40 -35 -30 -25 -20 -15 -10 -5 0 T (°C) Zeta Potential (mV) Effect of QXE on zeta for polyNIPA 20% MAA 1.00 g/L LCST 10 mV more stable than unimprinted polymer Increase in stability @ LCST Diagram of a 1,5-naphthyridine molecule being bound by polyNIPA/MAA Results The term “molecular recognition” describes specific interactions between two or more molecules that involve non-covalent bonding including hydrogen bonds, metal coordination, hydrophobic interactions, Van Der Walls interactions, π-π interactions, and electrostatic effects 1 . Molecular recognition is extremely common among biomolecules 1 . Biological receptors are highly selective to their complimentary substrates and are often stereoselective , but are very complex molecules that are difficult to synthesize. In addition each receptor is only useful for binding to unique substrates. A solution to this problem pursued in this project is a form of molecular imprinting using Uncrosslinked polymers of N-isopropylacrylamide and methacrylic acid to selectively bind analytes. Conclusions and Future Experiments References: (1) Nelson, D.; Cox, M. Principles of Biochemistry 5 th ed. New York. 2008. p. 88. (2) http://www.malvern.com/LabEng/technology/zeta_potential/zeta_potential_LDE.htm. (3) Malvern Instruments. Zetasizer Nano User Manual. Worcestershire, England. 2004. (4) Piletsky, S., Turner, A., Molecular Imprinting of Polymers, Landes Bioscience 2006 N H O OH O N-isopropylacrylam ide Methacrylicacid 80 m ol% 20 m ol% 0.4 mol% K 2 S 2 O 8 , pH 7 Phosphatebuffer(.1 M ) 24h, 65 C, N 2 ° N N N N N N 1,5-naphthyridine Quinoxaline Quinazoline TemplateM olecules In Concentrationsof 2,4,10 perbinding site Tem plate CO 2 H CO 2 H CO 2 H CO 2 H N H O OH O N-isopropylacrylamide Methacrylicacid N Vinylpyradine Polym erBackbone M onomer(80-94% ) Recognition M onomers(6-20% ) TargetM oleculesforSeparation Benzopyrazines Nitrophenols N N N N N N NO 2 OH NO 2 HO HO NO 2 analyte CO 2 H CO 2 H CO 2 H CO 2 H N N O O O O O O O O O O O O O O H 2 O H 2 O H 2 O H 2 O 15 20 25 30 35 40 45 50 55 60 65 -34 -30 -26 -22 -18 -14 -10 -6 -2 Zeta Potential (mV) poly(NIPA 90:10 MAA) 1.13 g/L pH=7.00 (synthesized in acetonitrile) Zeta Potential as a function of temperature T (°C) LCST much less apparent Lower zeta potential/stability How Partitioning Works - Synthesize imprinted polymer @ pH7 - Remove template molecule by dialysis - Time frame several hours to days - Experiments at 20, 45, and 65°C Imprinted polymer in Dialysis Tubing Dialysis Solvent pH 7 buffer 70% MeOH/25% H 2 O/5% HOAc pH 2 HCl Equilibrium Dialysis method: 1.00 mL Imprinted polymer on one side, 1.00 mL template solution (0.0001 M) on other side. Experiments lasted 24 h and were conducted at 28° and 45°C. Concentrations of template monitored by absorbance spectroscopy. Better Binding and selectivity at 45°C. Dialysis Cell Polymer (analyte) T = 28°C T = 45°C 2 BDSR QXE (QXE) ∆A 306 none ∆A 315 0.043 ∆A 306 none ∆A 315 0.070 10 BDSR QXE (QXE) ∆A 306 none ∆A 315 0.026 ∆A 306 none ∆A 315 0.049 2 BDSR QXE (QZE,QZE) ∆A 306 none ∆A 315 none ∆A 306 0.035 ∆A 315 0.053 10 BDSR QXE (QXE,QZE) ∆A 306 none ∆A 315 none ∆A 306 0.026 ∆A 315 0.037 0.0001 M QXE + 0.0001 M QZE Normalized QXE + QZE Solution after Equilibrium with 4 BDSR QZE imprinted polymer at 45°C ∆Absorbance during binding QZE response QXE response -Early experiments showed that the uncrosslinked template polymers are capable of re-binding target molecules. -Equilibrium dialysis experiments have shown selectivity for both QXE (10:1, 2:1 bdsr ) and QZE (4:1 bdsr) imprinted uncrosslinked polymers using a 0.0001 M standard solution of mixed QXE and QZE. -Selectivity was best for 4:1 bdsr polymers. -Temperature during dialysis has a major effect on binding with the best binding observed at temperatures just above the LCST (45C) with less binding or even no binding (Figure 8) at lower temperatures (28C). It does not seem to make a difference if the temperature is raised above 45C - -.New Experiment using RAFT polymerization to synthesize polymer, Vinylpyradine as recognition monomer, and Nitrophenols as target molecules. - Calculate relative selectivity ratios, or distribution ratios between the three polymers using equilibrium dialysis experiments and solutions of mixed analyte molecules. - Replicate the selective binding results I got for the two uncrosslinked polymers using similar binding experiments with a wider variety of crosslinked polymers. -Expand the molecular imprinting to amino acids and chiral molecules. It would be a major breakthrough to be able to selectively bind chiral molecules like in a real biochemical sensor. -Further characterization of the selective polymers by GPC and light scattering to determine the polydispersity and molecular weight. These will need to be known for this work to be potentially published Acknowledgement s: Dr. Rudolf Seitz and Research group John Csoros and Dr. Sterling Tomellini (5) O. Oktar, P. Caglar, W. R. Seitz, Chemical modulation of thermosensitive poly(N-isopropylacrylamide) microsphere swelling: a ne strategy for chemical sensing, Sensors and Actuators B: Chemical, Volume 104, Issue 2, 24 January 2005, Pages 179-185, ISSN 0925- 4005, DOI: 10.1016/j.snb.2004.04.033.
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Molecular Recognition Using Uncrosslinked Polymers of N-isopropylacrylamide. Alex Papantones. Results. Background. Molecular Recognition. How P artitioning Works. - PowerPoint PPT Presentation
Transcript of Molecular Recognition Using Uncrosslinked Polymers of N-isopropylacrylamide
Molecular Recognition Using Uncrosslinked Polymers of N-isopropylacrylamideAlex Papantones
N
N
N
N
N
N
Quinoxaline Quinazoline1,5-naphthyridine
λabs = 310.1 nm
λabs = 314.9 nm
λabs = 306.5 nm
Background
The molecules involved bind as “host” and “guest” molecules. The two molecules are complementary, and can form a coordination complex between each other that the host molecule cannot form with any other molecule. A good analogy is a keyhole and a key. The guest will easily bond to the host but any other guest molecule will not be able to bind to the host molecule at all.
Molecular Recognition
Polymerization detailsPolymers purified by dialysis- For this project concentrations of MAA were 0, 10, or 20 mol%- Reaction reaches completion in minutes- Free Radical synthesis, can use water or acetonitrile as solvent
Properties of polyNIPAPolyNIPA has a Lower Critical Solution Temperature (LCST) of 32°C. It is soluble in water below this temperature and will aggregate out of solution at higher temperatures. This process is reversible4.
Vials of 40 mg/mL polyNIPA 20% MAA in pH 7 HBES buffer at 65°C (left) and 20°C (right)
When the polymerization is carried out at high temperature in the presence of a template molecule, the molecules are surrounded by aggregated polymer imprinting that molecule into the polymer structure. Allowing the polymer to relax to the random coil state releases the template molecules during dialysis5.
Zeta Potential is the electrical potential between the dispersion medium and the stationary area of fluid attached to a dispersed particle. It can also be described as electrical potential in the interfacial double layer (DL) at the location of the slipping plane versus a point in the bulk fluid away from the interface. A value of 30 mV (+ or -) would be considered to be a highly charged, stable2
-There is a charge on the outer surfaces of polymer interacting with the solution-High zeta potential can tell us precisely at what temp LCST occursCan often give us clues as to the structure of a polymer aggregate-Measured by Zetasizer Nano instrument using Laser Doppler Velicometry3
25 30 35 40 45 50 55 60-35
-30
-25
-20
-15
-10
-5
0
poly(NIPA 80:20 MAA)
Temperature (*C)
Zeta
pot
entia
l (m
V)
15 20 25 30 35 40 45 50 55 60 65-50-45-40-35-30-25-20-15-10-50
T (°C)
Zeta
Pot
entia
l (m
V)
Effect of QXE on zeta for polyNIPA 20% MAA 1.00 g/L
LCST
10 mV more stable than unimprinted polymer
Increase in stability @ LCST
Diagram of a 1,5-naphthyridine molecule being bound by polyNIPA/MAA
ResultsThe term “molecular recognition” describes specific interactions between two or more molecules that involve non-covalent bonding including hydrogen bonds, metal coordination, hydrophobic interactions, Van Der Walls interactions, π-π interactions, and electrostatic effects1.
Molecular recognition is extremely common among biomolecules1. Biological receptors are highly selective to their complimentary substrates and are often stereoselective , but are very complex molecules that are difficult to synthesize. In addition each receptor is only useful for binding to unique substrates. A solution to this problem pursued in this project is a form of molecular imprinting using Uncrosslinked polymers of N-isopropylacrylamide and methacrylic acid to selectively bind analytes.
Conclusions and Future Experiments
References: (1) Nelson, D.; Cox, M. Principles of Biochemistry 5th ed. New York. 2008. p. 88. (2) http://www.malvern.com/LabEng/technology/zeta_potential/zeta_potential_LDE.htm. (3) Malvern Instruments. Zetasizer Nano User Manual. Worcestershire, England. 2004. (4) Piletsky, S., Turner, A., Molecular Imprinting of Polymers, Landes Bioscience 2006
NH
O
OH
O
N-isopropylacrylamide Methacrylic acid80 mol% 20 mol%
0.4 mol% K2S2O8, pH 7 Phosphate buffer (.1 M)
24h, 65 C, N2°
N
N
N
N
N
N
1,5-naphthyridine
Quinoxaline
Quinazoline
Template MoleculesIn Concentrations of 2,4,10 per binding site
Template
CO2H
CO2H
CO2H
CO2H
NH
O
OH
O
N-isopropylacrylamide Methacrylic acid
N
Vinylpyradine
Polymer BackboneMonomer (80-94%)
Recognition Monomers (6-20%)
Target Molecules for Separation
Benzopyrazines Nitrophenols
N
N
N
N
N
N
NO2
OH
NO2HO
HO
NO2
analyte
CO2H
CO2H
CO2H
CO2H
N
N
OO
O O
OO
O
O
O
O
O O
O
O
H2O
H2O
H2O
H2O 15 20 25 30 35 40 45 50 55 60 65-34-30-26-22-18-14-10-6-2
Zeta
Pot
entia
l (m
V)
poly(NIPA 90:10 MAA) 1.13 g/L pH=7.00 (synthesized in acetonitrile) Zeta Potential as a function of temperature
T (°C)
LCST much less apparent
Lower zeta potential/stability
How Partitioning Works- Synthesize imprinted polymer @ pH7 - Remove template molecule by dialysis - Time frame several hours to days- Experiments at 20, 45, and 65°C
Imprinted polymer in Dialysis Tubing
Dialysis SolventpH 7 buffer70% MeOH/25% H2O/5% HOAcpH 2 HCl
Equilibrium Dialysis method: 1.00 mL Imprinted polymer on one side, 1.00 mL template solution (0.0001 M) on other side. Experiments lasted 24 h and were conducted at 28° and 45°C.
Concentrations of template monitored by absorbance spectroscopy. Better Binding and selectivity at 45°C.
Dialysis Cell
Polymer (analyte) T = 28°C T = 45°C
2 BDSR QXE (QXE) ∆A306 none∆A315 0.043
∆A306 none∆A315 0.070
10 BDSR QXE (QXE) ∆A306 none∆A315 0.026
∆A306 none∆A315 0.049
2 BDSR QXE (QZE,QZE)
∆A306 none∆A315 none
∆A306 0.035∆A3150.053
10 BDSR QXE (QXE,QZE)
∆A306 none∆A315 none
∆A306 0.026∆A315 0.037
0.0001 M QXE + 0.0001 M QZENormalized QXE + QZESolution after Equilibrium with 4 BDSR QZE imprinted polymer at 45°C∆Absorbance during binding
QZE response QXE response
-Early experiments showed that the uncrosslinked template polymers are capable of re-binding target molecules. -Equilibrium dialysis experiments have shown selectivity for both QXE (10:1, 2:1 bdsr ) and QZE (4:1 bdsr) imprinted uncrosslinked polymers using a 0.0001 M standard solution of mixed QXE and QZE. -Selectivity was best for 4:1 bdsr polymers.-Temperature during dialysis has a major effect on binding with the best binding observed at temperatures just above the LCST (45C) with less binding or even no binding (Figure 8) at lower temperatures (28C). It does not seem to make a difference if the temperature is raised above 45C--.New Experiment using RAFT polymerization to synthesize polymer, Vinylpyradine as recognition monomer, and Nitrophenols as target molecules.
- Calculate relative selectivity ratios, or distribution ratios between the three polymers using equilibrium dialysis experiments and solutions of mixed analyte molecules.
- Replicate the selective binding results I got for the two uncrosslinked polymers using similar binding experiments with a wider variety of crosslinked polymers.
-Expand the molecular imprinting to amino acids and chiral molecules. It would be a major breakthrough to be able to selectively bind chiral molecules like in a real biochemical sensor. -Further characterization of the selective polymers by GPC and light scattering to determine the polydispersity and molecular weight. These will need to be known for this work to be potentially publishedAcknowledgements:
Dr. Rudolf Seitz and Research groupJohn Csoros and Dr. Sterling Tomellini
(5) O. Oktar, P. Caglar, W. R. Seitz, Chemical modulation of thermosensitive poly(N-isopropylacrylamide) microsphere swelling: a new strategy for chemical sensing, Sensors and Actuators B: Chemical, Volume 104, Issue 2, 24 January 2005, Pages 179-185, ISSN 0925-4005, DOI: 10.1016/j.snb.2004.04.033.
