Molecule-Sized Lock and Key
In this laboratory, the students will make a polymer than has been created in the presence of a template molecule (an adenine derivative, ethyl adenine-9-acetate). Removal of the template molecule leaves a cavity of complementary size, shape and functionality to the template molecule. This “imprinted polymer” then can be shown to have the ability to recognize and rebind the template molecule better than a control polymer that was made under the same conditions but without the template molecule. This binding experiment is carried out by equilibrating a known amount of the individual polymers with a solution of the template. The depletion or binding of the template is followed by UV spectroscopy. This process parallels many of the recognition processes used by biological molecules such as enzymes, antibodies, and cell receptors.
Student manual for the SEPA USC laboratory
Molecule-Sized Lock and Key
The ability of molecules to recognize one another is the basis for many of the important biological processes. For example, when you take a drug, it goes into you body and targets a specific set of protein receptors. Without this specificity, the drug might fit into every receptor and have many unintended side-effects. Another example is your immune system. A class of proteins called antibodies is able to recognize foreign molecules. The antibodies bind to the foreign molecules and assist in their removal from you body. If your antibodies are working properly, they only bind to the foreign molecules and not to the molecules that are supposed to be in our body.
The ability of molecules to recognize each other was hypothesized over 120 years ago. Emil Fischer in his 1894 Noble Prize in chemistry lecture compared the interaction of drugs with receptors in the body to the concept of a lock and key. Amazingly, Emil Fischer made this supposition well before the understanding of the structure of the protein receptors. Effectively (as the cartoon suggests), he knew only the existence and structure of various biomolecules (the keys) and he was able to postulate the existence of receptor proteins (the locks).
In this experiment, you will be making synthetic polymers that have the ability to selectively bind to an adenine derivative. Adenine is a common component of molecules. It is the “A” in many biologically important molecules such as ATP, ADP, SAM, FAD and NADH. You know about adenine because it is as one of the four bases in DNA. These examples demonstrate the importance of adenine as a recognition unit. You will be making a synthetic polymer that selectively binds ethyl adenine-9-acetate (EA9A). The polymer will be made using a technique called the molecular imprinting processes. This technique involves making a polymer in the presence of a template molecule (EA9A in this case) to produce a molecularly imprinted polymer (MIP).
Background
Molecularly imprinted polymers (MIPs) can be prepared in a single synthetic step to yield a polymer with easily tailored recognition properties. Although MIPs generally have much lower selectivity and binding affinities than normal biological recognition systems, they do possess superior thermal and chemical stability, in addition to their low cost and ease of preparation. Thus, MIPs have been used in a wide range of applications in place of antibodies in chiral separations, environmental toxin sensors, solid phase extraction, and catalysis.
A generalized imprinting strategy is outlined below (Scheme 1). The molecular template, EA9A, is added to a solution containing methacrylic acid (MAA), and the two form a hydrogen-bonded complex, which is ‘frozen’ in place by polymerization with a large excess of crosslinking agent, ethylene glycoldimethacrylate (EDMA) using a radical polymerization with azobisisobutyronitrile (AIBN) as the radical initiator. The MAA carboxylic acids become locked into place by the rigid matrix that has formed around the template. Removal of the EA9A template results in a polymer containing spaces that are selective for the EA9A template molecule. A solvent, called the porogen, also is added to the polymerization mixture to ensure that the crosslinked matrix has a porous structure that allows the template to diffuse into and out of the polymer. Polymers prepared in the absence of a porogen lack this porous structure and exhibit poorer recognition performance as a result.
Scheme 1. Non-covalent imprinting begins with the selected template (EA9A) complexing with a functional monomer (MAA). Crosslinking locks the monomers into the proper orientation around the template. Removal of the template results in selective binding sites for EA9A.
As a control, a second polymer is prepared under identical conditions in the absence of EA9A. The imprinted and control polymers are identical in composition and should differ only in their template-induced local conformations and structures. Differences in the recognition properties of the two polymers are then measures of the selective recognition properties of the imprinted polymer that arise from the specific cavities formed in the imprinting process. The assumption is that the imprinted polymer will contain more selective binding sites than the control polymer.
Hazards
This investigation requires the use of chemicals that are flammable (acetonitrile, methanol), toxic (methacrylic acid (CAS # 79-41-4), ethylene glycol dimethacrylate (CAS # 97-90-5), ethyl adenine-9-acetate (CAS # 25477-96-7), azobisisobutyronitrile (CAS # 78-67-1), acetonitrile (CAS # 75-05-8), methanol), corrosive (methacrylic acid) and that have a rancid odor (methacrylic acid). Appropriate safety procedures in the handling of these materials should be observed at all times.
Specify the two goals: 1) Make an imprinted and non-imprinted polymer for EA9A and 2) Be able to identify which polymer is which with binding experiments.
Student Directions
Caution! The chemicals used in this experiment are toxic and/or flammable. All liquids should be handled only in a functioning fume hood with proper safety equipment (apron, gloves and goggles) worn at all times.
Day One: Preparation and Processing of Polymers
1. In a 2 dram screw cap vial labeled “IMPRINTED”, obtain 2.5 mL of the prepolymerization mixture from your instructor and pipet it into the vial. Now weigh out 12.0 mg of the EA9A (the template) and add this to the mixture in the vial. Cap the vial and shake vigorously until the EA9A is completely dissolved. This will be the polymerization mixture for the imprinted polymer.
2. In a 2 dram screw cap vial labeled “CONTROL”, obtain 2.5 mL of the prepolymerization mixture from your instructor and pipet it into the vial. This will be the polymerization mixture for the non-imprinted polymer.
3. Place the vials in an oil bath at approximately 60 °C for one hour to allow the polymerization to proceed. Take care when submersing the vials in the oil to ensure the reaction mixture is below the level of the liquid while keeping the vial cap above the surface.
4. After one hour, carefully remove the vials from the oil bath and wipe the vial off with a dry paper towel. Wrap the IMPRINTED vial in a dry paper towel and use a hammer (or another blunt object) to strike it until the glass breaks. Carefully remove the polymer monolith within, taking care to brush off any residual glass fragments that may adhere to the polymer surface. Discard the glass, vial cap, and paper towel in the proper waste receptacle. Grind the polymer into a fine powder using a small mortar and pestle. When finished, the consistency should be that of flour or confectioner’s sugar.
5. Obtain a 40-ml blue-capped vial. Scrape the ground polymer into the vial and add enough methanol so that the vial is approximately half-full. Cap the vial and shake the mixture vigorously for 1-2 min and then allow the polymer to settle for ONLY (!) two minutes. Pour out the methanol (it will look cloudy, that’s ok) into a waste container. Add more methanol to the vial until it is half-full and shake for 1-2 minutes again, allow to settle, and decant (pour out) the methanol. Repeat with fresh methanol until you have completed 5 extractions total. Mark the vial with IMPRINTED and your initials and place it into the oven to dry until after lunch.
6. Rinse out the mortar and pestle with soapy water and wipe dry. Repeat the same procedure of wrapping, cracking, grinding, and extracting with methanol for the CONTROL polymer, making sure to write your initials on the vial before placing into the oven.
Day II: Testing and Analysis of Imprinted Polymers
1. Weigh out 100 mg of each of the polymers into separate 2-dram vials and label them appropriately.
2. Add 5-ml of the benzofurazan dye-tagged EA9A to each vial, cap and allow to shake for 10-15 minutes.
3. Allow the polymer to settle for 1 minute, then pipet 1-mL into the cuvette.
4. Measure and record the absorbance of the solution at 465 nm.
5. Repeat for the other polymer sample.
6. Measure and record the absorbance of the dye solution (not shaken with polymer).
Student Data Sheet
Beer’s Law: A = ebc
Line of Best Fit: c = ______A + ______
R2 = ______
e = ______(ask instructor)
3.0 Instructor Notes
Background Information
Imprinted polymers were investigated as early as the 1940’s but not studied systematically until Wulff et al. began pioneering research in this field in the early 1970’s. Covalent and non-covalent interactions between the template molecule and the functional monomer have been used to create imprinted polymers, with the former producing more homogeneous binding sites and the latter broader, heterogeneous distributions. Systems based on covalent interactions display binding characteristics that are well modeled by the Langmuir isotherm, while MIPs utilizing non-covalent forces are better modeled by the Freundlich isotherm. Since the form of the Freundlich (B = aFm) is a power function, when plotted as log B versus log F the binding isotherm can be fit well by a straight line. It has been observed that the “imprint effect”, as defined by the difference in bound analyte between the imprinted and control polymers at a given concentration is not constant over all concentration regimes, but rather improves under more dilute conditions. This is well illustrated in the log-log plot, where the binding isotherms of the imprinted and control polymers diverge at lower concentrations and converge at higher concentrations. This observation has lead some to believe that non-covalent MIPs will be most effective in applications which can tolerate or require lower analyte concentrations.
4.0 Chemical Hazards
All chemicals should be handled with care and protecting clothing such as a lab coat, shoes, protective gloves and eyewear should be worn at all times. All reactions should be carried out in a well-ventilated hood. All organic liquid wastes should be disposed of in an organic waste container. All organic solids should be disposed of in the solid organic waste container. Methacrylic acid should be kept in a cool, dry, dark location in a tightly sealed container. Keep it away from any incompatible materials such as strong oxidizing agents, ignition sources and untrained individuals. Methacrylic acid readily polymerizes in the presence of light, heat and oxygen, and also under the action of oxidizing agents such as peroxides. When heated, it can decompose into an acrid smoke with irritating fumes. Methacrylic acid should only be used under a well-ventilated hood. Ethylene glycol dimethacrylate should be stored in a tightly closed container also in a cool, dry, well-ventilated area away from incompatible substances such as strong acids, strong bases, strong oxidizing agents. EDMA can decompose into carbon monoxide, produce irritating and toxic fumes and gases. Acetonitrile is a highly flammable solvent. The toxic vapor is heavier than air and may travel considerable distance to source of ignition and flash back. Chronic inhalation and ingestion may cause effects similar to those of acute inhalation and ingestion. It also may be metabolized to cyanide which in turn acts by inhibiting cytochrome oxidase, impairing cellular respiration. Ethyl adenine-9-acetate (EA9A) should not be inhaled or allowed to come into contact with eyes, skin or clothing. EA9A should be stored in a tightly closed container in a cool and dry environment. Azobisisobutyronitrile is a free-radical initiator that may cause liver damage if inhaled; cover spilled AIBN with dry-lime, sand or soda ash and place in a sealed container.
Introduction to Imprinted Polymers
Pre-laboratory Quiz
1. In what ways are imprinted polymers similar to other molecular recognition systems? How are they different? (3 pts)
Suggested Response: Similarities: show selective binding to target analyte; rely on same interactions to bind template; binding properties are dependent upon pH, concentration, temperature. Differences: exist as insoluble crosslinked polymers; rigid binding sites as compared to enzymes; greater chemical and physical stability.
2. What are the names, chemical structures, and role of each reagent in the polymerization mixture? Draw the imprinting scheme. (5 pts)
3. How will the “imprinting effect” be measured in this investigation? Be specific. (2 pts)
Suggested Response: the imprinting effect will be measured by the difference of bound analyte at each given total concentration between the imprinted and control polymers. Or as the relative heights of the isotherms either in B vs. F or log (B) vs. log (F) form.
Introduction to Imprinted Polymers
Post-laboratory Questions
1. Consider the log-log isotherm you constructed. Do you think that the imprinted polymer performs better at lower or higher analyte concentrations? What is your evidence for this prediction? (3pts)
The log-log plot shows a divergence of the two isotherms at lower concentrations which is evidence for better performance whereas the B vs. F plot shows larger differences at higher concentrations which argues against this reasoning. In practice, it is the ratio of the bound/free concentrations (called the separation factor) between the two polymers that is usually considered the best measure of performance.
2. Graph B/F (y-axis) vs. B (x-axis) for the imprinted polymer. This is called a Scatchard plot and can be used to determine if all the binding sites in the polymer are equivalent (homogeneous) as well as the strength of the polymer-template interaction. If the Scatchard plot is well modeled by a straight line, the polymer can be considered homogeneous. If not, the polymer has sites of varying affinity and is thus considered heterogeneous. Did you prepare a homogeneous or heterogeneous imprinted polymer? (4 pts)