title: Semi-continuous, real-time monitoring of protein biomarker using a recyclable surface plasmon resonance sensor.
abstract: Although label-free immunosensors based on, for example, surface plasmon resonance (SPR) provide advantages of real-time monitoring of the analyte concentration, its application to routine clinical analysis in a semi-continuous manner is problematic because of the high cost of the sensor chip. The sensor chip is in most cases regenerated by employing an acidic pH. However, this causes gradual deterioration of the activity of the capture antibody immobilized on the sensor surface. To use sensor chips repeatedly, we investigated a novel surface modification method that enables regeneration of the sensor surface under mild conditions. We introduced a monoclonal antibody (anti-CBP Ab) that detects the conformational change in calcium binding protein (CBP) upon Ca2+binding (>1mM). To construct a regenerable SPR-based immunosensor, anti-CBP Ab was first immobilized on the sensor surface, and CBP conjugated to the capture antibody (specific for creatine kinase-MB isoform (CK-MB); CBP-CAb) then bound in the presence of Ca2+. A serum sample was mixed with the detection antibody to CK-MB, which generated an SPR signal proportional to the analyte concentration. After each analysis, the sensor surface was regenerated using medium (pH 7) without Ca2+, and then adding fresh CBP-CAb in the presence of Ca2+for the subsequent analysis. Analysis of multiple samples using the same sensor was reproducible at a rate >98.7%. The dose-response curve was linear for 1.75-500.75ng/mL CK-MB, with an acceptable coefficient of variation of <8.8%. The performance of the immunosensor showed a strong correlation with that of the Pathfast reference system (R2>96%), and exhibited analytical stability for 1 month. To our knowledge, this is the first report of a renewal of a sensor surface with fresh antibody after each analysis, providing high consistency in the assay during a long-term use (e.g., a month at least).
authors.affiliation: Department of Bio-Microsystem Technology, Korea University, 145 Anam-ro, Seongbuk-Gu, Seoul 02841, Republic of Korea.
authors.forename: Dong-Hyung
authors.initials: DH
authors.lastname: Kim
authors.affiliation: Department of Biomedical Laboratory Science, College of Health Science, Eulji University, Seongnam 13135, Republic of Korea.
authors.forename: Il-Hoon
authors.initials: IH
authors.lastname: Cho
authors.affiliation: Department of Bio-Microsystem Technology, Korea University, 145 Anam-ro, Seongbuk-Gu, Seoul 02841, Republic of Korea.
authors.forename: Ji-Na
authors.initials: JN
authors.lastname: Park
authors.affiliation: Department of Bio-Microsystem Technology, Korea University, 145 Anam-ro, Seongbuk-Gu, Seoul 02841, Republic of Korea.
authors.forename: Sung-Ho
authors.initials: SH
authors.lastname: Paek
authors.affiliation: Korea Research Institute of Standards and Science, P.O. Box 102, Yuseong, Taejon 34113, Republic of Korea.
authors.forename: Hyun-Mo
authors.initials: HM
authors.lastname: Cho
authors.affiliation: Department of Bio-Microsystem Technology, Korea University, 145 Anam-ro, Seongbuk-Gu, Seoul 02841, Republic of Korea; Department of Biotechnology, Korea University, 2511 Sejong-ro, Sejong 30019, Republic of Korea. Electronic address: shpaek@korea.ac.kr.
authors.forename: Se-Hwan
authors.initials: SH
authors.lastname: Paek
mesh_terms: D055910:Antibodies, Immobilized / Q000737:chemistry; D002118:Calcium / Q000737:chemistry; D002135:Calcium-Binding Proteins / Q000737:chemistry; D052279:Creatine Kinase, MB Form / Q000032:analysis / Q000097:blood*; D004867:Equipment Design; D006801:Humans; D007118:Immunoassay / Q000295:instrumentation / Q000379:methods*; D018796:Immunoconjugates / Q000737:chemistry; D057230:Limit of Detection; D011487:Protein Conformation; D015203:Reproducibility of Results; D020349:Surface Plasmon Resonance / Q000295:instrumentation / Q000379:methods*
chemical_list: D055910:Antibodies, Immobilized; D002135:Calcium-Binding Proteins; D018796:Immunoconjugates; D052279:Creatine Kinase, MB Form; D002118:Calcium
keywords: Acute myocardial infarction markers; Companion diagnostics; Conformation change-sensitive antibody; Label-free sensor regeneration; Semi-continuous immunosensor