Genetically Encoded Ratiometric pH Sensors for the Measurement of Intra- and Extracellular pH and Internalization Rates
pH-sensitive fluorescent proteins as genetically encoded pH sensors are promising tools for monitoring intra- and extracellular pH. However, there is a lack of ratiometric pH sensors, which offer a good dynamic range and can be purified and applied extracellularly to investigate uptake. In our study...
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2022-04-01
|
| Series: | Biosensors |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2079-6374/12/5/271 |
| _version_ | 1827670023236222976 |
|---|---|
| author | Lennard Karsten Lukas Goett-Zink Julian Schmitz Raimund Hoffrogge Alexander Grünberger Tilman Kottke Kristian M. Müller |
| author_facet | Lennard Karsten Lukas Goett-Zink Julian Schmitz Raimund Hoffrogge Alexander Grünberger Tilman Kottke Kristian M. Müller |
| author_sort | Lennard Karsten |
| collection | DOAJ |
| description | pH-sensitive fluorescent proteins as genetically encoded pH sensors are promising tools for monitoring intra- and extracellular pH. However, there is a lack of ratiometric pH sensors, which offer a good dynamic range and can be purified and applied extracellularly to investigate uptake. In our study, the bright fluorescent protein CoGFP_V0 was C-terminally fused to the ligand epidermal growth factor (EGF) and retained its dual-excitation and dual-emission properties as a purified protein. The tandem fluorescent variants EGF-CoGFP-mTagBFP2 (pK′ = 6.6) and EGF-CoGFP-mCRISPRed (pK′ = 6.1) revealed high dynamic ranges between pH 4.0 and 7.5. Using live-cell fluorescence microscopy, both pH sensor molecules permitted the conversion of fluorescence intensity ratios to detailed intracellular pH maps, which revealed pH gradients within endocytic vesicles. Additionally, extracellular binding of the pH sensors to cells expressing the EGF receptor (EGFR) enabled the tracking of pH shifts inside cultivation chambers of a microfluidic device. Furthermore, the dual-emission properties of EGF-CoGFP-mCRISPRed upon 488 nm excitation make this pH sensor a valuable tool for ratiometric flow cytometry. This high-throughput method allowed for the determination of internalization rates, which represents a promising kinetic parameter for the in vitro characterization of protein–drug conjugates in cancer therapy. |
| first_indexed | 2024-03-10T03:15:15Z |
| format | Article |
| id | doaj.art-1cce7ff657664e1f87e45761c5734923 |
| institution | Directory Open Access Journal |
| issn | 2079-6374 |
| language | English |
| last_indexed | 2024-03-10T03:15:15Z |
| publishDate | 2022-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biosensors |
| spelling | doaj.art-1cce7ff657664e1f87e45761c57349232023-11-23T10:15:04ZengMDPI AGBiosensors2079-63742022-04-0112527110.3390/bios12050271Genetically Encoded Ratiometric pH Sensors for the Measurement of Intra- and Extracellular pH and Internalization RatesLennard Karsten0Lukas Goett-Zink1Julian Schmitz2Raimund Hoffrogge3Alexander Grünberger4Tilman Kottke5Kristian M. Müller6Cellular and Molecular Biotechnology, Faculty of Technology, Bielefeld University, 33615 Bielefeld, GermanyBiophysical Chemistry and Diagnostics, Medical School OWL, Faculty of Chemistry, Bielefeld University, 33615 Bielefeld, GermanyMultiscale Bioengineering, Faculty of Technology, Bielefeld University, 33615 Bielefeld, GermanyCell Culture Technology, Faculty of Technology, Bielefeld University, 33615 Bielefeld, GermanyMultiscale Bioengineering, Faculty of Technology, Bielefeld University, 33615 Bielefeld, GermanyBiophysical Chemistry and Diagnostics, Medical School OWL, Faculty of Chemistry, Bielefeld University, 33615 Bielefeld, GermanyCellular and Molecular Biotechnology, Faculty of Technology, Bielefeld University, 33615 Bielefeld, GermanypH-sensitive fluorescent proteins as genetically encoded pH sensors are promising tools for monitoring intra- and extracellular pH. However, there is a lack of ratiometric pH sensors, which offer a good dynamic range and can be purified and applied extracellularly to investigate uptake. In our study, the bright fluorescent protein CoGFP_V0 was C-terminally fused to the ligand epidermal growth factor (EGF) and retained its dual-excitation and dual-emission properties as a purified protein. The tandem fluorescent variants EGF-CoGFP-mTagBFP2 (pK′ = 6.6) and EGF-CoGFP-mCRISPRed (pK′ = 6.1) revealed high dynamic ranges between pH 4.0 and 7.5. Using live-cell fluorescence microscopy, both pH sensor molecules permitted the conversion of fluorescence intensity ratios to detailed intracellular pH maps, which revealed pH gradients within endocytic vesicles. Additionally, extracellular binding of the pH sensors to cells expressing the EGF receptor (EGFR) enabled the tracking of pH shifts inside cultivation chambers of a microfluidic device. Furthermore, the dual-emission properties of EGF-CoGFP-mCRISPRed upon 488 nm excitation make this pH sensor a valuable tool for ratiometric flow cytometry. This high-throughput method allowed for the determination of internalization rates, which represents a promising kinetic parameter for the in vitro characterization of protein–drug conjugates in cancer therapy.https://www.mdpi.com/2079-6374/12/5/271pH sensorEGFratiometricGFPFRETdynamic range |
| spellingShingle | Lennard Karsten Lukas Goett-Zink Julian Schmitz Raimund Hoffrogge Alexander Grünberger Tilman Kottke Kristian M. Müller Genetically Encoded Ratiometric pH Sensors for the Measurement of Intra- and Extracellular pH and Internalization Rates Biosensors pH sensor EGF ratiometric GFP FRET dynamic range |
| title | Genetically Encoded Ratiometric pH Sensors for the Measurement of Intra- and Extracellular pH and Internalization Rates |
| title_full | Genetically Encoded Ratiometric pH Sensors for the Measurement of Intra- and Extracellular pH and Internalization Rates |
| title_fullStr | Genetically Encoded Ratiometric pH Sensors for the Measurement of Intra- and Extracellular pH and Internalization Rates |
| title_full_unstemmed | Genetically Encoded Ratiometric pH Sensors for the Measurement of Intra- and Extracellular pH and Internalization Rates |
| title_short | Genetically Encoded Ratiometric pH Sensors for the Measurement of Intra- and Extracellular pH and Internalization Rates |
| title_sort | genetically encoded ratiometric ph sensors for the measurement of intra and extracellular ph and internalization rates |
| topic | pH sensor EGF ratiometric GFP FRET dynamic range |
| url | https://www.mdpi.com/2079-6374/12/5/271 |
| work_keys_str_mv | AT lennardkarsten geneticallyencodedratiometricphsensorsforthemeasurementofintraandextracellularphandinternalizationrates AT lukasgoettzink geneticallyencodedratiometricphsensorsforthemeasurementofintraandextracellularphandinternalizationrates AT julianschmitz geneticallyencodedratiometricphsensorsforthemeasurementofintraandextracellularphandinternalizationrates AT raimundhoffrogge geneticallyencodedratiometricphsensorsforthemeasurementofintraandextracellularphandinternalizationrates AT alexandergrunberger geneticallyencodedratiometricphsensorsforthemeasurementofintraandextracellularphandinternalizationrates AT tilmankottke geneticallyencodedratiometricphsensorsforthemeasurementofintraandextracellularphandinternalizationrates AT kristianmmuller geneticallyencodedratiometricphsensorsforthemeasurementofintraandextracellularphandinternalizationrates |