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...
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| Format: | Article |
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MDPI AG
2020-06-01
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| Series: | Biosensors |
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| Online Access: | https://www.mdpi.com/2079-6374/10/6/71 |
| _version_ | 1797564262929924096 |
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| author | Evan Kazura Ray Mernaugh Franz Baudenbacher |
| author_facet | Evan Kazura Ray Mernaugh Franz Baudenbacher |
| author_sort | Evan Kazura |
| collection | DOAJ |
| description | 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 (<i>kcat</i>) and the inactivation factor (<i>InF</i>), using a known label enzyme amount (<i>Ea</i>). Over four discrete runs (<i>n</i> = 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. |
| first_indexed | 2024-03-10T18:54:54Z |
| format | Article |
| id | doaj.art-0ea8e52bf74647979b63c088fa7e791d |
| institution | Directory Open Access Journal |
| issn | 2079-6374 |
| language | English |
| last_indexed | 2024-03-10T18:54:54Z |
| publishDate | 2020-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biosensors |
| spelling | doaj.art-0ea8e52bf74647979b63c088fa7e791d2023-11-20T04:48:06ZengMDPI AGBiosensors2079-63742020-06-011067110.3390/bios10060071A Capillary-Perfused, Nanocalorimeter Platform for Thermometric Enzyme-Linked Immunosorbent Assay with Femtomole SensitivityEvan Kazura0Ray Mernaugh1Franz Baudenbacher2Department of Biomedical Engineering, Vanderbilt University, Nashville, TN 37235, USADepartment of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37235, USADepartment of Biomedical Engineering, Vanderbilt University, Nashville, TN 37235, USAEnzyme-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 (<i>kcat</i>) and the inactivation factor (<i>InF</i>), using a known label enzyme amount (<i>Ea</i>). Over four discrete runs (<i>n</i> = 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.https://www.mdpi.com/2079-6374/10/6/71microfabricated calorimeterELISAthermometric ELISAbiosensormodel-assisted signal analysis |
| spellingShingle | Evan Kazura Ray Mernaugh Franz Baudenbacher A Capillary-Perfused, Nanocalorimeter Platform for Thermometric Enzyme-Linked Immunosorbent Assay with Femtomole Sensitivity Biosensors microfabricated calorimeter ELISA thermometric ELISA biosensor model-assisted signal analysis |
| title | A Capillary-Perfused, Nanocalorimeter Platform for Thermometric Enzyme-Linked Immunosorbent Assay with Femtomole Sensitivity |
| title_full | A Capillary-Perfused, Nanocalorimeter Platform for Thermometric Enzyme-Linked Immunosorbent Assay with Femtomole Sensitivity |
| title_fullStr | A Capillary-Perfused, Nanocalorimeter Platform for Thermometric Enzyme-Linked Immunosorbent Assay with Femtomole Sensitivity |
| title_full_unstemmed | A Capillary-Perfused, Nanocalorimeter Platform for Thermometric Enzyme-Linked Immunosorbent Assay with Femtomole Sensitivity |
| title_short | A Capillary-Perfused, Nanocalorimeter Platform for Thermometric Enzyme-Linked Immunosorbent Assay with Femtomole Sensitivity |
| title_sort | capillary perfused nanocalorimeter platform for thermometric enzyme linked immunosorbent assay with femtomole sensitivity |
| topic | microfabricated calorimeter ELISA thermometric ELISA biosensor model-assisted signal analysis |
| url | https://www.mdpi.com/2079-6374/10/6/71 |
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