Colorimetric stack pad immunoassay for bacterial identification.
A new colorimetric immunoassay concept, utilizing conventional lateral flow membranes (e.g., conjugation, sample, absorption and nitrocellulose), were placed in a different configuration in a stacking manner, where the liquid sample that may contain the analyte diffuses from the bottom to the upper-most layer. The key element of this proprietary technology is a capture layer, where a nitrocellulose membrane is modified with the target analyte of interest, namely in this study target Escherichia coli. During the immunoassay operation, samples contaminated with the target bacteria will conjugate to their corresponding HRP-antibodies laying in wait and the immune-target measurand complex flows by capillarity towards the upper-most layer to generate a colorimetric signal (positive answer) through an enzymatic reaction. In target-free samples, previously immobilized target bacteria on the capture layer will prevent the HRP-labeled anti-target antibodies from migrating to the upper-most layer, where the enzymatic substrate lays in wait. After optimization, the sensitivity of this approach was found to be 1,000 folds higher than ELISAs (102cellsmL-1). The advantages of the stacked pad assay include: miniaturization, operational simplicity, fast response time (less than 5min), useful sensitivity.
School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, 639798, Singapore; Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel; NTU-HUJ-BGU CREATE Programme, 1 Create Way, Research Wing #02-06 to 08, 138602, Singapore.
Evgeni
E
Eltzov
School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, 639798, Singapore; Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel; NTU-HUJ-BGU CREATE Programme, 1 Create Way, Research Wing #02-06 to 08, 138602, Singapore; The National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel; The Ilse Katz Center for Meso and Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel. Electronic address: rsmarks@bgu.ac.il.
Robert S
RS
Marks
D055910:Antibodies, Immobilized / Q000737:chemistry; D015374:Biosensing Techniques / Q000191:economics / Q000295:instrumentation; D003101:Collodion / Q000737:chemistry; D003124:Colorimetry / Q000191:economics / Q000295:instrumentation*; D004784:Environmental Monitoring / Q000191:economics / Q000295:instrumentation; D004867:Equipment Design; D004926:Escherichia coli / Q000302:isolation & purification*; D004927:Escherichia coli Infections / Q000382:microbiology; D006801:Humans; D007118:Immunoassay / Q000191:economics / Q000295:instrumentation*; D057230:Limit of Detection; D008567:Membranes, Artificial; D045483:Rivers / Q000382:microbiology; D012623:Seawater / Q000382:microbiology; D014871:Water Microbiology
D055910:Antibodies, Immobilized; D008567:Membranes, Artificial; D003101:Collodion
Bacteria; Colorimetry; Immunoassay; Membranes; Miniaturization