Scanning electrochemical microscopy (SECM) was used to microfabricate and quantify diaphorase-pattemed glass surfaces. Deactivated circular and linear micropattems were produced at diaphorase-immobilized substrates by a localized surface reaction. The oxidation of Br~and Clã t a microelectrode generated a reactive species which deactivated the localized enzyme molecules at the substrate. The diaphorase-pattemed surfaces were characterized by SECM on the basis of detection of catalytic current of ferrocenylmethanol coupled with oxidation of NADH. The concentration of the immobilized diaphorase
The microfabrication and characterization of glass surfaces patterned with enzymes (diaphorase, horseradish peroxidase(HRP)) or antigen-antibodies (carcinoembryonic antigen (CEA), human chorionic gonadotropin (HCG) and human placental lactogen (HPL)) were studied using scanning electrochemical microscopy (SECM). Localized enzymes and antigen-antibody complexes with labeled enzymes were characterized on the basis of detection of catalytic current for ferrocenylmethanol by SECM. The SECM technique was extended to the enzyme-linked immunosorbent assay (ELISA). This method detects as low as ~ 10 4 CEA molecules in a single microspot. We also demonstrated a novel dual assay using microfabricated glass substrates with anti-HCG and anti-HPL microspots.Scanning electroehemical microscopy (SECM), a member of the scanning probe microscopies (SPM), uses an ultrarnicroelectrode as the probe to characterize the localized electrochemical properties of the surfaces (1-3). Although the lateral resolution of SECM is inferior to that of STM or AFM because it is difficult to fabricate a small probe microelectrode with atom-size radius, SECM has capabilities of detecting chemical reactions. In addition, it can induce chemical reactions in an extremely small volume. Using the unique properties of SECM, a single molecule (4) or a radical with a short lifetime (5) was recently detected.We report here the SECM characterization of glass surfaces micropatterned with enzymes or antigen-antibodies. Patterned enzymes or enzyme-labeled complexes of antigen and antibody were viewed by SECM based on monitoring catalytic reactions of the patterned enzymes. We have drawn lines and spots of diaphorase, horseradish peroxidase (HRP), HRP-labeled antigen-antibody complexes with carcinoembryonic antigen (CEA), human chorionic gonadotropin (HCG) and human placental lactogen (HPL) on glass substrates by a motor-driven glass capillary pen. The dimension of the patterns can be controlled by changing the outer tip radius of the pen. It is also possible to create a pattern with more than two proteins in a very small area with μπι dimensions. We also demonstrate here a dual assay of HCG and HPL at a photofabricated glass substrate.
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