title: Dual Signal Amplification Electrochemical Biosensor for Monitoring the Activity and Inhibition of the Alzheimer's Related Protease β-Secretase.
abstract: The protease BACE1 (the β-site amyloid precursor protein cleaving enzyme 1) catalyzes the first step in the synthesis of β-amyloids (Aβ), peptides that accumulate in the brain in Alzheimer's disease (AD). Measurement of BACE1 activity is important for the development of BACE1 inhibitors to slow or stop AD. To measure BACE1 cleavage of the electrode-immobilized substrate peptide, we developed a redox-generating hydroxyapatite (HAP) probe which generates electrochemical current by reaction of the nanoparticle with molybdate (MoO42-). The probe combines alkaline phosphatase (ALP) for dual signal amplification and Aβ antibody to bind the probe to the immobilized peptide substrate on the surface of the electrode. We measured the activity of BACE1 at concentrations ranging from 0.25 to 100 U/mL. The use of the dual-signal HAP-ALP probe increased the signal by an order of magnitude compared to HAP-only probe, enabling detection limits as low as 0.1 U/mL. To measure the inhibition of BACE1 activity, the BACE1 inhibitor OM99-2 was added to 25 U/mL of BACE1 in concentrations ranging from 5 to 150 nM. The observed detection limit of inhibition is 10 nM of OM99-2. These results demonstrate the capabilities of this novel biosensor to measure BACE1 activity and inhibitors of BACE1 activity. To the best of our knowledge this is the first report that reaction of HAP nanoparticles with molybdate can generate electrochemical current. This dual signal amplification strategy can be extended to other electrochemical assays and adapted for wide applications.
authors.affiliation: College of Chemistry and Chemical Engineering, Central South University , Changsha, 410083, China.|College of Chemistry and Chemical Engineering, Qufu Normal University , Qufu, Shandong 273165, China.
authors.forename: Fengli
authors.initials: F
authors.lastname: Qu
authors.affiliation: College of Chemistry and Chemical Engineering, Central South University , Changsha, 410083, China.
authors.forename: Minghui
authors.initials: M
authors.lastname: Yang
authors.affiliation: National Cancer Institute, National Institutes of Health , Rockville, Maryland 20850, United States.
authors.forename: Avraham
authors.initials: A
authors.lastname: Rasooly
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry; D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors* / Q000097:blood* / Q000737:chemistry / Q000276:immunology; D000906:Antibodies / Q000276:immunology; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors* / Q000097:blood* / Q000737:chemistry / Q000276:immunology; D015374:Biosensing Techniques / Q000379:methods*; D017886:Durapatite / Q000737:chemistry*; D055664:Electrochemical Techniques / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods; D006801:Humans; D057230:Limit of Detection; D008982:Molybdenum / Q000737:chemistry; D053758:Nanoparticles / Q000737:chemistry*; D009842:Oligopeptides / Q000737:chemistry; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry; D015203:Reproducibility of Results
chemical_list: D000906:Antibodies; C414614:OM99-2; D009842:Oligopeptides; D010455:Peptides; C044659:molybdate; D008982:Molybdenum; D017886:Durapatite; D000469:Alkaline Phosphatase; D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human