Label-free electrical determination of trypsin activity by a silicon-on-insulator based thin film resistor.
A silicon-on-insulator (SOI) based thin film resistor is employed for the label-free determination of enzymatic activity. We demonstrate that enzymes, which cleave biological polyelectrolyte substrates, can be detected by the sensor. As an application, we consider the serine endopeptidase trypsin, which cleaves poly-L-lysine (PLL). We show that PLL adsorbs quasi-irreversibly to the sensor and is digested by trypsin directly at the sensor surface. The created PLL fragments are released into the bulk solution due to kinetic reasons. This results in a measurable change of the surface potential allowing for the determination of trypsin concentrations down to 50 ng mL(-1). Chymotrypsin is a similar endopeptidase with a different specificity, which cleaves PLL with a lower efficiency as compared to trypsin. The activity of trypsin is analyzed quantitatively employing a kinetic model for enzyme-catalyzed surface reactions. Moreover, we have demonstrated the specific inactivation of trypsin by a serine protease inhibitor, which covalently binds to the active site of the enzyme.
Neff
Petra A
PA
Lehrstuhl für Biophysik-E22, Technische Universität München, Germany.
Serr
Andreas
A
Wunderlich
Bernhard K
BK
Bausch
Andreas R
AR
D015374:Biosensing Techniques; D002384:Catalysis; D002627:Chemistry, Physical / Q000379:methods*; D002918:Chymotrypsin / Q000737:chemistry*; D004563:Electrochemistry / Q000379:methods; D010450:Endopeptidases / Q000737:chemistry; D007700:Kinetics; D011107:Polylysine / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D012825:Silicon / Q000737:chemistry*; D012822:Silicon Dioxide / Q000737:chemistry; D013499:Surface Properties; D013997:Time Factors; D014357:Trypsin / Q000737:chemistry*
D011108:Polymers; D011107:Polylysine; D012822:Silicon Dioxide; D010450:Endopeptidases; D002918:Chymotrypsin; D014357:Trypsin; D012825:Silicon