David Michaelis and Dr. Paul Savage, Chemistry and Biochemistry

In recent years it has become critical to detect and monitor concentrations of heavy metal ions in the environment. Cadmium and other heavy metals have detrimental effects to human health. The main difficulty of sensing these ions is selectively detecting the ions in the presence of other naturally present metal ions such as calcium, zinc, and copper. Fluorescent molecular sensors provide an ideal solution to the problem. These organic sensors can be constructed to selectively bind with target metal ions. While many such sensors exist today, little work has been done to incorporate such sensors into portable sensing devices.

In response to this problem, a method was devised for attaching said sensors onto glass surfaces. Through the formation of self assembled monolayers, a uniform layer of molecular sensors is applied to a glass surface for use in portable sensing devices. Once attached, it is crucial that the molecule maintains its ability to interact with metal ions in solution. The purpose of this project is to determine effect that binding a molecular sensor to a solid surface has on the sensor’s ability to detect metal ions. A molecular sensor specific for cadmium was chosen as a test sensor.

The first step in this project was to determine the reproducibility of bonding sensors to glass via self-assembled monolayers. A problem arose when directly attaching the sensor to the glass surface did not provide sufficient density of the sensor on the surface. Thus, it was impossible to obtain a measurable response to the metals by the sensor. In response to this problem, a monolayer of polymer (poly(methyl vinyl ether-alt-maleic anhydride)) was first formed on the glass surface which provided additional sites where the sensor could attach. To test the reproducibility of this method, 4 sets of 3 slides were synthesized following similar procedures. The surfaces were then compared using UV-visual spectroscopy, which showed similar properties in each of the slides among the four runs. This procedure provided a higher density of molecular sensors per unit area, producing a larger fluorescent response by each glass slide.

Our next goal was to determine how the sensor’s response to metal ions changed when bound to a solid surface. For this purpose, a flow cell was constructed which would allow solutions containing metal ions to flow continuously over the slide. In this manner, the sensors sensitivity to cadmium, as well as its selectivity to cadmium over other metal ions was measured. Aqueous solutions of cadmium mixed individually with zinc, calcium, and copper in varying concentrations were used in the experiment. A buffer solution of 10 mM sodium acetate at a PH of 5.2 was used to measure baseline fluorescence and for preparation of all metal ion solutions. Fluorescence measurements were performed with a Jobin Yvon Horiba FluoroMax-3 spectrofluorometer using a xenon lamp as the excitation source as was initially planned.

While performing test runs, a gradual increase in the baseline fluorescence of the sensor was noticed. Later, it was discovered that air bubbles forming in the flow cell were the cause of the increasing baseline. To solve the problem, the PH buffer and metal ion solutions were first sonicated to remove dissolved carbon dioxide, and then degassed using argon gas. These procedures minimized the formation of air bubbles on the glass slide and eliminated the increase in the baseline fluorescence.

In testing the glass-bound sensor, similar results were obtained to those recorded for the sensor in its unbound state . In the presence of cadmium, even at 10 µM concentrations, a large increase in fluorescence was measured. Zinc also produced an increase in fluorescence, about 2/3 the magnitude of the increase seen with cadmium. Copper on the other had the effect of dramatically decreasing or quenching the baseline fluorescence. These results are consistent with the response recorded with the sensor in its free state. The consistency in the response by the sensor indicates that the ability of the sensor to bind with metal ions in solution is unaffected when the sensor is bound to a solid surface.

In studies involving a competition between cadmium and other metal ions, various results were recorded. At similar concentrations of cadmium and zinc, only a small additional increase in fluorescence was measured over the fluorescence of cadmium alone. This indicates that the sensor maintained its selectivity for cadmium over zinc. At higher concentrations of zinc, however, fluorescence increased above what its fluorescence with cadmium alone. This may indicate that once sensor molecules were saturated with cadmium, left over sensors were able to bind with zinc and increase fluorescence. In competitive experiments with copper and cadmium, a decrease in fluorescence was recorded. The decrease in fluorescence due to the copper/cadmium solutions was not as dramatic as when copper was introduced alone, suggesting that copper does not completely override the sensor’s ability to interact with cadmium.

While interaction with cadmium and other metal ions caused a large change in fluorescence, returning fluorescence to its baseline level proved difficult at first. Because of the high affinity of these sensors for metal ions, it was difficult wash the metal off the sensor. In initial tests, it took as long as 1 hour for fluorescence magnitudes to return to pre-test levels, indicating that all metal ions had been removed from the sensor. In solution to this problem, a buffer of higher acidity (PH 4.2) was pulsed through the cell for about 30 seconds after each measurement was taken. This had the effect of temporarily protonating the active sites on the sensor used for binding to the metal ions, thus allowing the metals to escape the sensor. In this manner, baseline fluorescence levels were reached in 3-4 minutes, rather than an hour as seen previously.

While these experiments show that binding a fluorescent sensor to a solid surface has little effect on the properties of the sensor molecule, additional work is needed to construct a portable metal ion sensor. The photophysical properties of our chosen compound and its complexes with cadmium, zinc, and copper provide valuable information on measuring fluorescent response due to individual ligand-metal interactions. Because the fluorescent response to each ion is inherently different, time based fluorescence spectroscopy may make it possible to selectively measure the fluorescence change due to each metal ion. Through time-based fluorescent studies, selective detection of cadmium in the presence of other metal ions via portable sensing devices may become a reality.