Technology
Bacteria at tip of electrode make it sensitive to arginine
Biochemical electrode uses living cells If an enzyme works, why not the organism that makes it? That reasoning has led to development of a biochemical electrode probe that uses living cells rather than isolated enzymes to make it biologically selective. Developed by graduate student Robert K. Kobos and Dr. Garry A. Rechnitz of the chemistry department of the State University of New York, Buffalo, the probe is based on a potentiometric elec trode sensitive to ammonia. Used with the electrode is a strain of bacteria that con verts the amino acid arginine into am monia. Together they form an electrode that gives a sensitive, selective, and effi cient response to arginine over a wide range of concentrations. Traditionally, one type of biochemical electrode probe couples an enzyme with a miniature ion- or gas-selective electrode. The enzyme reacts only with a very nar row range of molecules in a reaction that yields a molecule that the electrode can measure along with other products. The electrode contributes extreme sensitivity so that a very small sample can be ana lyzed, and the enzyme provides biological selectivity. Such systems are being used increasingly in biological monitoring and other medical uses (C&EN, Jan. 27,1975, page 29). Ordinarily the enzyme for this type of electrode must be identified, isolated, and stored in a form that keeps it active in order to be used, Rechnitz explains. If the entire microorganism that makes an en zyme could be used instead, the enzyme would not need to be isolated or even specifically identified. Such an approach also would take advantage of the optimi zation of reaction conditions for a specific reaction that has taken place within some cells through centuries of evolution. And some intact cells are much more stable than isolated enzymes. They even may be able to be cultured so that an electrode would regenerate itself when not in use. Sometimes several cofactors are nec essary for an enzyme reaction to occur, and getting them all present along with the enzyme at the tip of an electrode by traditional chemical means can be diffi cult, Rechnitz explains. A living cell pro vides a complete package of the enzyme and cofactors. And it can be used some what empirically even when the exact biochemical steps taking place within the cell have not been worked out. The electrode developed by Kobos and Rechnitz is a fairly simple one to prepare. The researchers smeared cultured Streptococcus faecium cells on the gaspermeable membrane of a conventional ammonia-sensing electrode and covered the bacteria layer with a cellophane dial ysis membrane to keep the bacteria in place. In their experiments this electrode
Dialysis membrane
Streptococcus faecium
gives a nearly Nernstian response to ar ginine for a concentration range from 10~ 2 M to 10~5 M at physiological pH. It has at least a 100-fold selectivity for arginine over other materials tested, including urea and the amino acids lysine and histidine. The bacteria electrode continues to be sensitive with very little loss of activity for at least three weeks, the chemists find, making its useful lifetime comparable to conventional enzyme electrodes. And preliminary experiments suggest that the activity of the electrode may be regener ated by returning it to the nutrient solu
Gas-permeable membrane
tion used to culture the bacteria when the electrode begins to run down. "We now see an entire heirarchy of possible mediating materials ranging from single enzymes on up in complexity to intact living organisms," Rechnitz says. "What is really novel about these elec trodes is that we are going toward a sys tem already optimized by nature through evolution." Rechnitz hopes in particular to use intact bacteria to make sensitive electrodes for molecules that cannot now be detected using electrochemical probes because an appropriate enzyme has not been identified or isolated. D
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Oct. 25, 1976 C&EN
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