The Chemical Educator, Vol. 12, No. 6, Published on Web 10/7/2007, 10.1333/s00897072086a, © 2007 The Chemical Educator

Construction of a Simple Myoglobin-Based Optical Biosensor

Todd A. Wells

Department of Chemistry and Biochemistry, University of Denver, Denver, CO, 80208, towells@du.edu

Received November 13, 2006. Accepted March 30, 2007.

Abstract: Optical biosensors exploit the theoretical and instrumental approaches of spectroscopy and the unique properties of biological molecules. We present here the design and construction of a simple inexpensive optical biosensor. The sensor is based on an LED colorimeter and polymer entrapped myoglobin. Myoglobin’s absorbance maximum is sensitive to ligand binding. Moreover, the overlap of its absorbance spectrum with the emission of a green LED increases with cyanide binding. The sensors response was linear at least in the range of 50 to 200 mM cyanide. The experiment is appropriate for introductory chemistry, environmental chemistry, instrumental analysis, and biochemistry students. It was initially developed for a summer program with advanced high school students.

Introduction

Biosensors are finding use in increasingly broader ranges of applications. For example, their use has dramatically increased in clinical diagnosis and biomedicine, pharmaceutical and drug analysis, pollution control and monitoring, and detection of industrial and toxic gases; however, a review of the literature did not yield any simple, inexpensive method for conveying an understanding of this technology. In this experiment, students constructed a very simple optical biosensor. The sensor originates from an LED/photoresistor-based colorimeter [1] coupled with immobilized myoglobin. The construction and application of this biosensor is suitable for introductory chemistry, environmental chemistry, instrumental analysis, and biochemistry courses that explore the basic components and applications of spectrometers.

Metalloporphyrin-containing proteins such as myoglobin are intensely colored and their absorbance wavelength maxima are sensitive to the interaction with ligands such as CN–, CO, O2, N3–, and F–. In this work, we focused on cyanide specifically. According to the Toxics Release Inventory, cyanide compound releases totaled about 1.5 million lb from 1987 to 1993 [2]. These releases were chiefly from steel mills and metal heat-treating industries. The cyanide ion can rapidly combine with iron in cytochrome a3 or cytochrome oxidase. Cyanide also has a high affinity for the ferric iron in methemoglobin. The immediately-dangerous-to-life-and-health (IDLH) concentration of hydrogen cyanide is 50 ppm. The oral LD50 for sodium and potassium cyanide are about 100 and 200 mg/kg, respectively. The Maximum Contaminant Level Goals (MCLG) for cyanide is 0.2 ppm (mg/L) [3].

Biosensors are analytical devices that use biological interactions with molecules to provide qualitative and quantitative results that are specific for the molecule being analyzed. These analytical devices incorporate a biological material or a biomimic (e.g., tissue, microorganisms, organelles, cell receptors, enzymes, antibodies, nucleic acids, etc.), intimately associated with or integrated to a transducing system [4, 5]. The advantage of using a biological sensing element is their remarkable ability to distinguish between the analyte of interest and similar substances. Another advantage is that biosensors are quick at providing information and they are simple to use. Their simplicity results from the incorporation of the biological element and transducer into one unit so the analysis can be essentially completed in one step. Figure 1 depicts the general arrangement of a biosensor and its components. There are a variety of transducing systems that can be used such as optical, electrochemical, thermometric, piezoelectric, or magnetic systems.

The science of biosensors has evolved significantly in the last several years. The first biosensors, called enzyme electrodes, were developed to detect glucose [4]. Research efforts for developing glucose biosensors have opened the way for the application of biosensors to other molecules. Other types of biosensors include the construction of an optical biosensor for the detection of insulin [6] and a fluorometric biosensor for the detection of fermentation activity [7].

Experimental

Myoglobin was entrapped by mixing it to a final concentration of 1 mg/mL in a 10% acrylamide/1.67% bisacrylamide and 10 mM Tris-Cl pH 7.4, giving a final volume of 15 ml. After thorough mixing, 0.05 ml of 10 % (w/v) ammonium persulfate and 0.01 ml of TEMED were added. The mixture was then divided amongst several small, ~2.0-mL, glass vials with rubber septa (0.50–0.75 mL each). The septa had been modified by punching a hole in each just large enough to insert an LED (a rubber stopper can be substituted for the cap). Two small needles were inserted in the septa next to the LED. Green LEDs (Radio Shack, VF = 2.1 V) were used as the light source. The LED voltage was maintained at 2 V using a 15-turn, 10 kΩ trimpot, (potentiometer) and a 9-V battery. The detector was a Radio Shack, CdS, photoresistor. A photoresistor was glued to the bottom of each vial by using a thin layer of five-minute epoxy. The photoresistor was connected to the 9-V battery in series with a 1 kΩ resistor. Because the resistors in series form a voltage divider, the voltage can be measured across the fixed resistor. Voltage is proportional to the light intensity impingent on the photoresistor.

The entire assembly was then wrapped with black electrical tape. The “dark” voltage (Vdark) was measured with the LED off. The cell was filled with water via a syringe connected to one of the needles. With the LED lit, the voltage measured is the 100% transmittance (Vreference). The water was removed from the cell and the cell was then filled with the cyanide containing sample solutions. The voltage was again measured, Vsample. Absorbance was calculated using the following relationship:

Figure 1. General layout of a biosensor.

Figure 2. A simple myoglobin based biosensor.

Figure 3. Comparison between the absorbance spectra of myoglobin and myoglobin with cyanide and the emission spectrum of the green LED.

Figure 4. The myoglobin based biosensor with increasing cyanide concentrations.

            

The UV/vis absorbance spectroscopy of myoglobin and the emission measurement of the LED were carried out using an Ocean Optics (Dunedin, FL) USB2000 spectrophotometer.

Hazards. Cyanide is toxic and contact with acid liberates poisonous gas. Acrylamide is a suspect carcinogen and a neurotoxin. In order to minimize handling of these substances, it is recommended that all sodium cyanide and acrylamide solutions be pre-made and provided to everyone but upper-level students. The students should wear gloves and safety goggles during the experiment. The cyanide containing solutions should be collected and oxidized prior to disposal. This is accomplished by adjusting the pH to about 10 followed by treatment with excess commercial bleach (sodium hypochlorite). The solution pH is then neutralized.

Results and Discussion

In the construction of a biosensor there are several considerations that must be made in the selection of a suitable biological element, a suitable immobilization method, and in the selection of a suitable transducer [8]. The selection of the biological element involves searching for one that binds efficiently to the analyte or analytes being studied. Here, we have chosen myoglobin as the biological element. The immobilization method should be carefully chosen so that the biological element remains functional. A common method of immobilization is entrapment in a hydrogel (polyacrylamide) as was used here. Finally, the transducer should be chosen so that the chemical interaction of the biological element with the sample is converted into an electrical signal that can beprocessed and displayed. Because the absorbance of myoglobin red-shifts upon cyanide binding, a simple LED-based colorimeter was chosen as the transducer.

The green LED used for the biosensor had a maximum emission wavelength of 560 nm, which overlaps significantly more with the cyanide adduct of myoglobin than the unliganded Fe(III) form (Figure 3). Figure 4 is a plot of absorbance versus cyanide concentration. The sensor has a linear response in the concentration range examined. Care must be taken to allow the sample solution to equilibrate with the polymer matrix. Faster equilibration can be achieved by using a thinner polyacrylamide-myoglobin element; however, it may be necessary to increase the myoglobin concentration.

A single needle inserted through the septum may be used instead of separate inlet and outlet needle. It is recommended that liquid transfer be accomplished via a needle and notby cap removal becaause this may cause a change in the distance between the LED and photoresistor. The light level measured by the photoresistor does vary with the separation distance.

Conclusion

The work presented here entails construction of a low-cost, simple biosensor that illustrates all of the components of an optical biosensor. It also shows the elementary concepts of electronics, spectroscopy, and ligand binding. The biosensor can be constructed and a standard curve generated in a three-hour laboratory. Success rates for construction of a functional biosensor are very high. The most frequent problem has been failure of the acrylamide to polymerize. The other common problem observed is instability in the measured voltage, which can be addressed by allowing the hydrogel sufficient time to equilibrate with the sample.Prior to assembly of the complete biosensor, it is helpful for students to build the colorimeter separately using a protoboard. Furthermore, the entrapped myoglobin can be formed in a small disposable glass test tube. The tube can then be placed between the LED and the photoresistor and samples added on top of the polymer.

Supporting Materials. Detailed student handouts for the construction of the optical biosensor are available in a Zip file (http://dx.doi.org/10.1333/s00897072086a).

References and Notes

1.       Gordon, J.; James, A.; Harman, S.; Weiss, K. J. Chem. Educ. 2002, 79 (8), 1005–1006.

2.       EPA 816-F-03-016, 2006. 

3.       EPA, Title 40 Protection of Environment, Part 141 National Primary Drinking Water Regulations; pp 364–365.

4.       Eggins, B. Biosensors: An Introduction; John Wiley and Sons: New York, NY; 1996, pp 3–6.

5.       Kissinger, P. T.Biosens. Bioelectron 2005, 20, 2512–2516.

6.       Disley, D. M.; Morril, P. R.; Sproule, K; Lowe, C. R. Biosens. Bioelectron. 1999, 14, 481–493.

7.       Li, J. K.; Asali, E. C.; Horvath, J. J. Biotechnol. Prog. 1991, 7, 21–27.

8.       Eggins, B. Biosensors: An Introduction; John Wiley and Sons: New York, NY; 1996, pp 8–10.