| A biosensor chip aimed for the evaluation of the electrochemical effects of immobilization of enzymes was designed, fabricated and tested. The chip includes sensor structures for the potentiometric, amperometric and impedometric detection of biological reactions catalyzed by enzymes immobilized in the same manner, on the same substrate. The chip and its packaging were designed to allow different immobilization schemes to be used without chip design modification.; A platinum micro-electrochemical electrode is used for the amperometric and impedometric detection. The potentiometric sensor is a differential pair, consisting of an ion sensitive field effect transistor (ISFET) and an enzyme field effect transistor (ENFET). The n-channel ISFET and ENFET are fabricated in a p-well, with junction isolation from the solution. A photodefinable polyimide is used as an encapsulatant and to define the area of the platinum electrode. The sensor wafer is packaged by bonding a micromachined package wafer, which has holes etched to expose the active areas of the sensors and bond pads, on the surface of the sensor wafer. The bonded sensor die is mounted on a ceramic substrate to bring the leads out. The bond wires and pads and the edges of the die are encapsulated with a polyurethane encapsulant.; The biosensor chip was evaluated using the penicillin-penicillinase (EC 3.5.2.6) system. The penicillinase was immobilized by physical adsorption on a Langmuir-Blodgett (LB) film. The LB film, deposited from a pure water subphase, consisted of multilayers of pure docosanoic acid, or a few base layers of 22-tricosenoic acid and then docosanoic acid. A technique for the patterning of the LB films by a modified lift-off process was developed, so that the LB film only covers the active areas of the sensors.; The penicillinase catalyzed reaction hydrolyses the penicillin into penicillinoic acid, which changes the local pH of the solution. The ENFET/ISFET differential potentiometric sensor showed a detection limit of 5 {dollar}times{dollar} 10{dollar}sp4{dollar} M penicillin-G in a 0.63 mM phosphate buffer solution. At high penicillin concentration, the penicillin-G overwhelmed the buffer in raising the pH of the solution, reducing the activity of penicillinase. The amperometric response showed a small penicillin-G concentration dependence because detecting hydrogen ions at a platinum electrode is very difficult, since the hydrolysis of water generates very large background noise. The impedometric detection showed a concentration dependent response in the phase angle of the electrode impedance at about 4 kHz, when the electrode was biased at +0.4 V versus a standard calomel electrode. |
