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A Capillary-Perfused, Nanocalorimeter Platform for Thermometric Enzyme-Linked Immunosorbent Assay with Femtomole Sensitivity
Enzyme-catalyzed chemical reactions produce heat. We developed an enclosed, capillary-perfused nanocalorimeter platform for thermometric enzyme-linked immunosorbent assay (TELISA). We used catalase as enzymes to model the thermal characteristics of the micromachined calorimeter. Model-assisted signa...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7344772/ https://www.ncbi.nlm.nih.gov/pubmed/32599750 http://dx.doi.org/10.3390/bios10060071 |
Sumario: | Enzyme-catalyzed chemical reactions produce heat. We developed an enclosed, capillary-perfused nanocalorimeter platform for thermometric enzyme-linked immunosorbent assay (TELISA). We used catalase as enzymes to model the thermal characteristics of the micromachined calorimeter. Model-assisted signal analysis was used to calibrate the nanocalorimeter and to determine reagent diffusion, enzyme kinetics, and enzyme concentration. The model-simulated signal closely followed the experimental signal after selecting for the enzyme turnover rate (kcat) and the inactivation factor (InF), using a known label enzyme amount (Ea). Over four discrete runs (n = 4), the minimized model root mean square error (RMSE) returned 1.80 ± 0.54 fmol for the 1.5 fmol experiments, and 1.04 ± 0.37 fmol for the 1 fmol experiments. Determination of enzyme parameters through calibration is a necessary step to track changing enzyme kinetic characteristics and improves on previous methods to determine label enzyme amounts on the calorimeter platform. The results obtained using model-system signal analysis for calibration led to significantly improved nanocalorimeter platform performance. |
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