A red-shifted Bioluminescence Resonance Energy Transfer (BRET) biosensing system for rapid measurement of plasmin activity in human plasma
Proteases are key signalling molecules for many physiological processes and their dysregulation is implicated in the progression of a range of diseases. Sensitive methods to measure protease activities in complex biological samples are critical for rapid disease diagnoses. The proteolytic activity o...
Ausführliche Beschreibung
Gespeichert in:
| Autor*in: |
Weihs, Felix [verfasserIn] |
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| Format: |
E-Artikel |
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| Sprache: |
Englisch |
| Erschienen: |
2020transfer abstract |
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| Schlagwörter: |
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| Umfang: |
10 |
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| Übergeordnetes Werk: |
Enthalten in: Neuro-Brucellosis - Gouider, R. ELSEVIER, 2015, an international journal devoted to all branches of analytical chemistry, Amsterdam |
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| Übergeordnetes Werk: |
volume:1102 ; year:2020 ; day:15 ; month:03 ; pages:99-108 ; extent:10 |
| Links: |
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| DOI / URN: |
10.1016/j.aca.2019.12.044 |
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ELV049329111 |
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| 245 | 1 | 0 | |a A red-shifted Bioluminescence Resonance Energy Transfer (BRET) biosensing system for rapid measurement of plasmin activity in human plasma |
| 264 | 1 | |c 2020transfer abstract | |
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| 520 | |a Proteases are key signalling molecules for many physiological processes and their dysregulation is implicated in the progression of a range of diseases. Sensitive methods to measure protease activities in complex biological samples are critical for rapid disease diagnoses. The proteolytic activity of plasmin reflects the fibrinolysis state of blood and its deregulation can indicate pathologies such as bleeding events. While Bioluminescence Resonance Energy Transfer (BRET) is a powerful and sensitive method for the detection of protease activity, the commonly applied blue-shifted BRET2 system, consisting of the Renilla luciferase Rluc2 and the large-stokes shift fluorescent protein GFP2, suffers from light absorption and light scattering in human plasma samples. To address this challenge, we developed a red-shifted BRET-based plasmin sensor by substituting BRET2 with the BRET6 system. BRET6 is composed of the red-shifted RLuc8.6 luciferase linked to the red light emitting fluorescent protein TurboFP635. The BRET6 biosensor exhibited 3-fold less light absorption in plasma samples compared to the BRET2 sensor leading to an up to a 5-fold increase in sensitivity for plasmin detection in plasma. The limits of detection for plasmin were determined to be 11.90 nM in 7.5% (v/v) plasma with a 10 min assay which enables biologically relevant plasmin activities of thrombolytic therapies to be detected. While a colorigenic plasmin activity assay achieved a similar detection limit of 10.91 nM in 7.5% (v/v) human plasma, it required a 2 h incubation period. The BRET6 sensor described here is faster and more specific than the colorigenic assay as it did not respond to unspiked human plasma samples. | ||
| 520 | |a Proteases are key signalling molecules for many physiological processes and their dysregulation is implicated in the progression of a range of diseases. Sensitive methods to measure protease activities in complex biological samples are critical for rapid disease diagnoses. The proteolytic activity of plasmin reflects the fibrinolysis state of blood and its deregulation can indicate pathologies such as bleeding events. While Bioluminescence Resonance Energy Transfer (BRET) is a powerful and sensitive method for the detection of protease activity, the commonly applied blue-shifted BRET2 system, consisting of the Renilla luciferase Rluc2 and the large-stokes shift fluorescent protein GFP2, suffers from light absorption and light scattering in human plasma samples. To address this challenge, we developed a red-shifted BRET-based plasmin sensor by substituting BRET2 with the BRET6 system. BRET6 is composed of the red-shifted RLuc8.6 luciferase linked to the red light emitting fluorescent protein TurboFP635. The BRET6 biosensor exhibited 3-fold less light absorption in plasma samples compared to the BRET2 sensor leading to an up to a 5-fold increase in sensitivity for plasmin detection in plasma. The limits of detection for plasmin were determined to be 11.90 nM in 7.5% (v/v) plasma with a 10 min assay which enables biologically relevant plasmin activities of thrombolytic therapies to be detected. While a colorigenic plasmin activity assay achieved a similar detection limit of 10.91 nM in 7.5% (v/v) human plasma, it required a 2 h incubation period. The BRET6 sensor described here is faster and more specific than the colorigenic assay as it did not respond to unspiked human plasma samples. | ||
| 650 | 7 | |a Bioluminescence |2 Elsevier | |
| 650 | 7 | |a Human plasma |2 Elsevier | |
| 650 | 7 | |a Protease activity |2 Elsevier | |
| 650 | 7 | |a Fibrinolysis |2 Elsevier | |
| 650 | 7 | |a Bioluminescence resonance energy transfer |2 Elsevier | |
| 650 | 7 | |a Plasmin |2 Elsevier | |
| 700 | 1 | |a Peh, Alex |4 oth | |
| 700 | 1 | |a Dacres, Helen |4 oth | |
| 773 | 0 | 8 | |i Enthalten in |n Elsevier Science |a Gouider, R. ELSEVIER |t Neuro-Brucellosis |d 2015 |d an international journal devoted to all branches of analytical chemistry |g Amsterdam |w (DE-627)ELV013501887 |
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10.1016/j.aca.2019.12.044 doi /cbs_pica/cbs_olc/import_discovery/elsevier/einzuspielen/GBV00000000000904.pica (DE-627)ELV049329111 (ELSEVIER)S0003-2670(19)31500-4 DE-627 ger DE-627 rakwb eng 610 VZ 540 VZ 35.10 bkl Weihs, Felix verfasserin aut A red-shifted Bioluminescence Resonance Energy Transfer (BRET) biosensing system for rapid measurement of plasmin activity in human plasma 2020transfer abstract 10 nicht spezifiziert zzz rdacontent nicht spezifiziert z rdamedia nicht spezifiziert zu rdacarrier Proteases are key signalling molecules for many physiological processes and their dysregulation is implicated in the progression of a range of diseases. Sensitive methods to measure protease activities in complex biological samples are critical for rapid disease diagnoses. The proteolytic activity of plasmin reflects the fibrinolysis state of blood and its deregulation can indicate pathologies such as bleeding events. While Bioluminescence Resonance Energy Transfer (BRET) is a powerful and sensitive method for the detection of protease activity, the commonly applied blue-shifted BRET2 system, consisting of the Renilla luciferase Rluc2 and the large-stokes shift fluorescent protein GFP2, suffers from light absorption and light scattering in human plasma samples. To address this challenge, we developed a red-shifted BRET-based plasmin sensor by substituting BRET2 with the BRET6 system. BRET6 is composed of the red-shifted RLuc8.6 luciferase linked to the red light emitting fluorescent protein TurboFP635. The BRET6 biosensor exhibited 3-fold less light absorption in plasma samples compared to the BRET2 sensor leading to an up to a 5-fold increase in sensitivity for plasmin detection in plasma. The limits of detection for plasmin were determined to be 11.90 nM in 7.5% (v/v) plasma with a 10 min assay which enables biologically relevant plasmin activities of thrombolytic therapies to be detected. While a colorigenic plasmin activity assay achieved a similar detection limit of 10.91 nM in 7.5% (v/v) human plasma, it required a 2 h incubation period. The BRET6 sensor described here is faster and more specific than the colorigenic assay as it did not respond to unspiked human plasma samples. Proteases are key signalling molecules for many physiological processes and their dysregulation is implicated in the progression of a range of diseases. Sensitive methods to measure protease activities in complex biological samples are critical for rapid disease diagnoses. The proteolytic activity of plasmin reflects the fibrinolysis state of blood and its deregulation can indicate pathologies such as bleeding events. While Bioluminescence Resonance Energy Transfer (BRET) is a powerful and sensitive method for the detection of protease activity, the commonly applied blue-shifted BRET2 system, consisting of the Renilla luciferase Rluc2 and the large-stokes shift fluorescent protein GFP2, suffers from light absorption and light scattering in human plasma samples. To address this challenge, we developed a red-shifted BRET-based plasmin sensor by substituting BRET2 with the BRET6 system. BRET6 is composed of the red-shifted RLuc8.6 luciferase linked to the red light emitting fluorescent protein TurboFP635. The BRET6 biosensor exhibited 3-fold less light absorption in plasma samples compared to the BRET2 sensor leading to an up to a 5-fold increase in sensitivity for plasmin detection in plasma. The limits of detection for plasmin were determined to be 11.90 nM in 7.5% (v/v) plasma with a 10 min assay which enables biologically relevant plasmin activities of thrombolytic therapies to be detected. While a colorigenic plasmin activity assay achieved a similar detection limit of 10.91 nM in 7.5% (v/v) human plasma, it required a 2 h incubation period. The BRET6 sensor described here is faster and more specific than the colorigenic assay as it did not respond to unspiked human plasma samples. Bioluminescence Elsevier Human plasma Elsevier Protease activity Elsevier Fibrinolysis Elsevier Bioluminescence resonance energy transfer Elsevier Plasmin Elsevier Peh, Alex oth Dacres, Helen oth Enthalten in Elsevier Science Gouider, R. ELSEVIER Neuro-Brucellosis 2015 an international journal devoted to all branches of analytical chemistry Amsterdam (DE-627)ELV013501887 volume:1102 year:2020 day:15 month:03 pages:99-108 extent:10 https://doi.org/10.1016/j.aca.2019.12.044 Volltext GBV_USEFLAG_U GBV_ELV SYSFLAG_U SSG-OLC-PHA GBV_ILN_40 GBV_ILN_120 35.10 Physikalische Chemie: Allgemeines VZ AR 1102 2020 15 0315 99-108 10 |
| spelling |
10.1016/j.aca.2019.12.044 doi /cbs_pica/cbs_olc/import_discovery/elsevier/einzuspielen/GBV00000000000904.pica (DE-627)ELV049329111 (ELSEVIER)S0003-2670(19)31500-4 DE-627 ger DE-627 rakwb eng 610 VZ 540 VZ 35.10 bkl Weihs, Felix verfasserin aut A red-shifted Bioluminescence Resonance Energy Transfer (BRET) biosensing system for rapid measurement of plasmin activity in human plasma 2020transfer abstract 10 nicht spezifiziert zzz rdacontent nicht spezifiziert z rdamedia nicht spezifiziert zu rdacarrier Proteases are key signalling molecules for many physiological processes and their dysregulation is implicated in the progression of a range of diseases. Sensitive methods to measure protease activities in complex biological samples are critical for rapid disease diagnoses. The proteolytic activity of plasmin reflects the fibrinolysis state of blood and its deregulation can indicate pathologies such as bleeding events. While Bioluminescence Resonance Energy Transfer (BRET) is a powerful and sensitive method for the detection of protease activity, the commonly applied blue-shifted BRET2 system, consisting of the Renilla luciferase Rluc2 and the large-stokes shift fluorescent protein GFP2, suffers from light absorption and light scattering in human plasma samples. To address this challenge, we developed a red-shifted BRET-based plasmin sensor by substituting BRET2 with the BRET6 system. BRET6 is composed of the red-shifted RLuc8.6 luciferase linked to the red light emitting fluorescent protein TurboFP635. The BRET6 biosensor exhibited 3-fold less light absorption in plasma samples compared to the BRET2 sensor leading to an up to a 5-fold increase in sensitivity for plasmin detection in plasma. The limits of detection for plasmin were determined to be 11.90 nM in 7.5% (v/v) plasma with a 10 min assay which enables biologically relevant plasmin activities of thrombolytic therapies to be detected. While a colorigenic plasmin activity assay achieved a similar detection limit of 10.91 nM in 7.5% (v/v) human plasma, it required a 2 h incubation period. The BRET6 sensor described here is faster and more specific than the colorigenic assay as it did not respond to unspiked human plasma samples. Proteases are key signalling molecules for many physiological processes and their dysregulation is implicated in the progression of a range of diseases. Sensitive methods to measure protease activities in complex biological samples are critical for rapid disease diagnoses. The proteolytic activity of plasmin reflects the fibrinolysis state of blood and its deregulation can indicate pathologies such as bleeding events. While Bioluminescence Resonance Energy Transfer (BRET) is a powerful and sensitive method for the detection of protease activity, the commonly applied blue-shifted BRET2 system, consisting of the Renilla luciferase Rluc2 and the large-stokes shift fluorescent protein GFP2, suffers from light absorption and light scattering in human plasma samples. To address this challenge, we developed a red-shifted BRET-based plasmin sensor by substituting BRET2 with the BRET6 system. BRET6 is composed of the red-shifted RLuc8.6 luciferase linked to the red light emitting fluorescent protein TurboFP635. The BRET6 biosensor exhibited 3-fold less light absorption in plasma samples compared to the BRET2 sensor leading to an up to a 5-fold increase in sensitivity for plasmin detection in plasma. The limits of detection for plasmin were determined to be 11.90 nM in 7.5% (v/v) plasma with a 10 min assay which enables biologically relevant plasmin activities of thrombolytic therapies to be detected. While a colorigenic plasmin activity assay achieved a similar detection limit of 10.91 nM in 7.5% (v/v) human plasma, it required a 2 h incubation period. The BRET6 sensor described here is faster and more specific than the colorigenic assay as it did not respond to unspiked human plasma samples. Bioluminescence Elsevier Human plasma Elsevier Protease activity Elsevier Fibrinolysis Elsevier Bioluminescence resonance energy transfer Elsevier Plasmin Elsevier Peh, Alex oth Dacres, Helen oth Enthalten in Elsevier Science Gouider, R. ELSEVIER Neuro-Brucellosis 2015 an international journal devoted to all branches of analytical chemistry Amsterdam (DE-627)ELV013501887 volume:1102 year:2020 day:15 month:03 pages:99-108 extent:10 https://doi.org/10.1016/j.aca.2019.12.044 Volltext GBV_USEFLAG_U GBV_ELV SYSFLAG_U SSG-OLC-PHA GBV_ILN_40 GBV_ILN_120 35.10 Physikalische Chemie: Allgemeines VZ AR 1102 2020 15 0315 99-108 10 |
| allfields_unstemmed |
10.1016/j.aca.2019.12.044 doi /cbs_pica/cbs_olc/import_discovery/elsevier/einzuspielen/GBV00000000000904.pica (DE-627)ELV049329111 (ELSEVIER)S0003-2670(19)31500-4 DE-627 ger DE-627 rakwb eng 610 VZ 540 VZ 35.10 bkl Weihs, Felix verfasserin aut A red-shifted Bioluminescence Resonance Energy Transfer (BRET) biosensing system for rapid measurement of plasmin activity in human plasma 2020transfer abstract 10 nicht spezifiziert zzz rdacontent nicht spezifiziert z rdamedia nicht spezifiziert zu rdacarrier Proteases are key signalling molecules for many physiological processes and their dysregulation is implicated in the progression of a range of diseases. Sensitive methods to measure protease activities in complex biological samples are critical for rapid disease diagnoses. The proteolytic activity of plasmin reflects the fibrinolysis state of blood and its deregulation can indicate pathologies such as bleeding events. While Bioluminescence Resonance Energy Transfer (BRET) is a powerful and sensitive method for the detection of protease activity, the commonly applied blue-shifted BRET2 system, consisting of the Renilla luciferase Rluc2 and the large-stokes shift fluorescent protein GFP2, suffers from light absorption and light scattering in human plasma samples. To address this challenge, we developed a red-shifted BRET-based plasmin sensor by substituting BRET2 with the BRET6 system. BRET6 is composed of the red-shifted RLuc8.6 luciferase linked to the red light emitting fluorescent protein TurboFP635. The BRET6 biosensor exhibited 3-fold less light absorption in plasma samples compared to the BRET2 sensor leading to an up to a 5-fold increase in sensitivity for plasmin detection in plasma. The limits of detection for plasmin were determined to be 11.90 nM in 7.5% (v/v) plasma with a 10 min assay which enables biologically relevant plasmin activities of thrombolytic therapies to be detected. While a colorigenic plasmin activity assay achieved a similar detection limit of 10.91 nM in 7.5% (v/v) human plasma, it required a 2 h incubation period. The BRET6 sensor described here is faster and more specific than the colorigenic assay as it did not respond to unspiked human plasma samples. Proteases are key signalling molecules for many physiological processes and their dysregulation is implicated in the progression of a range of diseases. Sensitive methods to measure protease activities in complex biological samples are critical for rapid disease diagnoses. The proteolytic activity of plasmin reflects the fibrinolysis state of blood and its deregulation can indicate pathologies such as bleeding events. While Bioluminescence Resonance Energy Transfer (BRET) is a powerful and sensitive method for the detection of protease activity, the commonly applied blue-shifted BRET2 system, consisting of the Renilla luciferase Rluc2 and the large-stokes shift fluorescent protein GFP2, suffers from light absorption and light scattering in human plasma samples. To address this challenge, we developed a red-shifted BRET-based plasmin sensor by substituting BRET2 with the BRET6 system. BRET6 is composed of the red-shifted RLuc8.6 luciferase linked to the red light emitting fluorescent protein TurboFP635. The BRET6 biosensor exhibited 3-fold less light absorption in plasma samples compared to the BRET2 sensor leading to an up to a 5-fold increase in sensitivity for plasmin detection in plasma. The limits of detection for plasmin were determined to be 11.90 nM in 7.5% (v/v) plasma with a 10 min assay which enables biologically relevant plasmin activities of thrombolytic therapies to be detected. While a colorigenic plasmin activity assay achieved a similar detection limit of 10.91 nM in 7.5% (v/v) human plasma, it required a 2 h incubation period. The BRET6 sensor described here is faster and more specific than the colorigenic assay as it did not respond to unspiked human plasma samples. Bioluminescence Elsevier Human plasma Elsevier Protease activity Elsevier Fibrinolysis Elsevier Bioluminescence resonance energy transfer Elsevier Plasmin Elsevier Peh, Alex oth Dacres, Helen oth Enthalten in Elsevier Science Gouider, R. ELSEVIER Neuro-Brucellosis 2015 an international journal devoted to all branches of analytical chemistry Amsterdam (DE-627)ELV013501887 volume:1102 year:2020 day:15 month:03 pages:99-108 extent:10 https://doi.org/10.1016/j.aca.2019.12.044 Volltext GBV_USEFLAG_U GBV_ELV SYSFLAG_U SSG-OLC-PHA GBV_ILN_40 GBV_ILN_120 35.10 Physikalische Chemie: Allgemeines VZ AR 1102 2020 15 0315 99-108 10 |
| allfieldsGer |
10.1016/j.aca.2019.12.044 doi /cbs_pica/cbs_olc/import_discovery/elsevier/einzuspielen/GBV00000000000904.pica (DE-627)ELV049329111 (ELSEVIER)S0003-2670(19)31500-4 DE-627 ger DE-627 rakwb eng 610 VZ 540 VZ 35.10 bkl Weihs, Felix verfasserin aut A red-shifted Bioluminescence Resonance Energy Transfer (BRET) biosensing system for rapid measurement of plasmin activity in human plasma 2020transfer abstract 10 nicht spezifiziert zzz rdacontent nicht spezifiziert z rdamedia nicht spezifiziert zu rdacarrier Proteases are key signalling molecules for many physiological processes and their dysregulation is implicated in the progression of a range of diseases. Sensitive methods to measure protease activities in complex biological samples are critical for rapid disease diagnoses. The proteolytic activity of plasmin reflects the fibrinolysis state of blood and its deregulation can indicate pathologies such as bleeding events. While Bioluminescence Resonance Energy Transfer (BRET) is a powerful and sensitive method for the detection of protease activity, the commonly applied blue-shifted BRET2 system, consisting of the Renilla luciferase Rluc2 and the large-stokes shift fluorescent protein GFP2, suffers from light absorption and light scattering in human plasma samples. To address this challenge, we developed a red-shifted BRET-based plasmin sensor by substituting BRET2 with the BRET6 system. BRET6 is composed of the red-shifted RLuc8.6 luciferase linked to the red light emitting fluorescent protein TurboFP635. The BRET6 biosensor exhibited 3-fold less light absorption in plasma samples compared to the BRET2 sensor leading to an up to a 5-fold increase in sensitivity for plasmin detection in plasma. The limits of detection for plasmin were determined to be 11.90 nM in 7.5% (v/v) plasma with a 10 min assay which enables biologically relevant plasmin activities of thrombolytic therapies to be detected. While a colorigenic plasmin activity assay achieved a similar detection limit of 10.91 nM in 7.5% (v/v) human plasma, it required a 2 h incubation period. The BRET6 sensor described here is faster and more specific than the colorigenic assay as it did not respond to unspiked human plasma samples. Proteases are key signalling molecules for many physiological processes and their dysregulation is implicated in the progression of a range of diseases. Sensitive methods to measure protease activities in complex biological samples are critical for rapid disease diagnoses. The proteolytic activity of plasmin reflects the fibrinolysis state of blood and its deregulation can indicate pathologies such as bleeding events. While Bioluminescence Resonance Energy Transfer (BRET) is a powerful and sensitive method for the detection of protease activity, the commonly applied blue-shifted BRET2 system, consisting of the Renilla luciferase Rluc2 and the large-stokes shift fluorescent protein GFP2, suffers from light absorption and light scattering in human plasma samples. To address this challenge, we developed a red-shifted BRET-based plasmin sensor by substituting BRET2 with the BRET6 system. BRET6 is composed of the red-shifted RLuc8.6 luciferase linked to the red light emitting fluorescent protein TurboFP635. The BRET6 biosensor exhibited 3-fold less light absorption in plasma samples compared to the BRET2 sensor leading to an up to a 5-fold increase in sensitivity for plasmin detection in plasma. The limits of detection for plasmin were determined to be 11.90 nM in 7.5% (v/v) plasma with a 10 min assay which enables biologically relevant plasmin activities of thrombolytic therapies to be detected. While a colorigenic plasmin activity assay achieved a similar detection limit of 10.91 nM in 7.5% (v/v) human plasma, it required a 2 h incubation period. The BRET6 sensor described here is faster and more specific than the colorigenic assay as it did not respond to unspiked human plasma samples. Bioluminescence Elsevier Human plasma Elsevier Protease activity Elsevier Fibrinolysis Elsevier Bioluminescence resonance energy transfer Elsevier Plasmin Elsevier Peh, Alex oth Dacres, Helen oth Enthalten in Elsevier Science Gouider, R. ELSEVIER Neuro-Brucellosis 2015 an international journal devoted to all branches of analytical chemistry Amsterdam (DE-627)ELV013501887 volume:1102 year:2020 day:15 month:03 pages:99-108 extent:10 https://doi.org/10.1016/j.aca.2019.12.044 Volltext GBV_USEFLAG_U GBV_ELV SYSFLAG_U SSG-OLC-PHA GBV_ILN_40 GBV_ILN_120 35.10 Physikalische Chemie: Allgemeines VZ AR 1102 2020 15 0315 99-108 10 |
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10.1016/j.aca.2019.12.044 doi /cbs_pica/cbs_olc/import_discovery/elsevier/einzuspielen/GBV00000000000904.pica (DE-627)ELV049329111 (ELSEVIER)S0003-2670(19)31500-4 DE-627 ger DE-627 rakwb eng 610 VZ 540 VZ 35.10 bkl Weihs, Felix verfasserin aut A red-shifted Bioluminescence Resonance Energy Transfer (BRET) biosensing system for rapid measurement of plasmin activity in human plasma 2020transfer abstract 10 nicht spezifiziert zzz rdacontent nicht spezifiziert z rdamedia nicht spezifiziert zu rdacarrier Proteases are key signalling molecules for many physiological processes and their dysregulation is implicated in the progression of a range of diseases. Sensitive methods to measure protease activities in complex biological samples are critical for rapid disease diagnoses. The proteolytic activity of plasmin reflects the fibrinolysis state of blood and its deregulation can indicate pathologies such as bleeding events. While Bioluminescence Resonance Energy Transfer (BRET) is a powerful and sensitive method for the detection of protease activity, the commonly applied blue-shifted BRET2 system, consisting of the Renilla luciferase Rluc2 and the large-stokes shift fluorescent protein GFP2, suffers from light absorption and light scattering in human plasma samples. To address this challenge, we developed a red-shifted BRET-based plasmin sensor by substituting BRET2 with the BRET6 system. BRET6 is composed of the red-shifted RLuc8.6 luciferase linked to the red light emitting fluorescent protein TurboFP635. The BRET6 biosensor exhibited 3-fold less light absorption in plasma samples compared to the BRET2 sensor leading to an up to a 5-fold increase in sensitivity for plasmin detection in plasma. The limits of detection for plasmin were determined to be 11.90 nM in 7.5% (v/v) plasma with a 10 min assay which enables biologically relevant plasmin activities of thrombolytic therapies to be detected. While a colorigenic plasmin activity assay achieved a similar detection limit of 10.91 nM in 7.5% (v/v) human plasma, it required a 2 h incubation period. The BRET6 sensor described here is faster and more specific than the colorigenic assay as it did not respond to unspiked human plasma samples. Proteases are key signalling molecules for many physiological processes and their dysregulation is implicated in the progression of a range of diseases. Sensitive methods to measure protease activities in complex biological samples are critical for rapid disease diagnoses. The proteolytic activity of plasmin reflects the fibrinolysis state of blood and its deregulation can indicate pathologies such as bleeding events. While Bioluminescence Resonance Energy Transfer (BRET) is a powerful and sensitive method for the detection of protease activity, the commonly applied blue-shifted BRET2 system, consisting of the Renilla luciferase Rluc2 and the large-stokes shift fluorescent protein GFP2, suffers from light absorption and light scattering in human plasma samples. To address this challenge, we developed a red-shifted BRET-based plasmin sensor by substituting BRET2 with the BRET6 system. BRET6 is composed of the red-shifted RLuc8.6 luciferase linked to the red light emitting fluorescent protein TurboFP635. The BRET6 biosensor exhibited 3-fold less light absorption in plasma samples compared to the BRET2 sensor leading to an up to a 5-fold increase in sensitivity for plasmin detection in plasma. The limits of detection for plasmin were determined to be 11.90 nM in 7.5% (v/v) plasma with a 10 min assay which enables biologically relevant plasmin activities of thrombolytic therapies to be detected. While a colorigenic plasmin activity assay achieved a similar detection limit of 10.91 nM in 7.5% (v/v) human plasma, it required a 2 h incubation period. The BRET6 sensor described here is faster and more specific than the colorigenic assay as it did not respond to unspiked human plasma samples. Bioluminescence Elsevier Human plasma Elsevier Protease activity Elsevier Fibrinolysis Elsevier Bioluminescence resonance energy transfer Elsevier Plasmin Elsevier Peh, Alex oth Dacres, Helen oth Enthalten in Elsevier Science Gouider, R. ELSEVIER Neuro-Brucellosis 2015 an international journal devoted to all branches of analytical chemistry Amsterdam (DE-627)ELV013501887 volume:1102 year:2020 day:15 month:03 pages:99-108 extent:10 https://doi.org/10.1016/j.aca.2019.12.044 Volltext GBV_USEFLAG_U GBV_ELV SYSFLAG_U SSG-OLC-PHA GBV_ILN_40 GBV_ILN_120 35.10 Physikalische Chemie: Allgemeines VZ AR 1102 2020 15 0315 99-108 10 |
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a red-shifted bioluminescence resonance energy transfer (bret) biosensing system for rapid measurement of plasmin activity in human plasma |
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A red-shifted Bioluminescence Resonance Energy Transfer (BRET) biosensing system for rapid measurement of plasmin activity in human plasma |
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Proteases are key signalling molecules for many physiological processes and their dysregulation is implicated in the progression of a range of diseases. Sensitive methods to measure protease activities in complex biological samples are critical for rapid disease diagnoses. The proteolytic activity of plasmin reflects the fibrinolysis state of blood and its deregulation can indicate pathologies such as bleeding events. While Bioluminescence Resonance Energy Transfer (BRET) is a powerful and sensitive method for the detection of protease activity, the commonly applied blue-shifted BRET2 system, consisting of the Renilla luciferase Rluc2 and the large-stokes shift fluorescent protein GFP2, suffers from light absorption and light scattering in human plasma samples. To address this challenge, we developed a red-shifted BRET-based plasmin sensor by substituting BRET2 with the BRET6 system. BRET6 is composed of the red-shifted RLuc8.6 luciferase linked to the red light emitting fluorescent protein TurboFP635. The BRET6 biosensor exhibited 3-fold less light absorption in plasma samples compared to the BRET2 sensor leading to an up to a 5-fold increase in sensitivity for plasmin detection in plasma. The limits of detection for plasmin were determined to be 11.90 nM in 7.5% (v/v) plasma with a 10 min assay which enables biologically relevant plasmin activities of thrombolytic therapies to be detected. While a colorigenic plasmin activity assay achieved a similar detection limit of 10.91 nM in 7.5% (v/v) human plasma, it required a 2 h incubation period. The BRET6 sensor described here is faster and more specific than the colorigenic assay as it did not respond to unspiked human plasma samples. |
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Proteases are key signalling molecules for many physiological processes and their dysregulation is implicated in the progression of a range of diseases. Sensitive methods to measure protease activities in complex biological samples are critical for rapid disease diagnoses. The proteolytic activity of plasmin reflects the fibrinolysis state of blood and its deregulation can indicate pathologies such as bleeding events. While Bioluminescence Resonance Energy Transfer (BRET) is a powerful and sensitive method for the detection of protease activity, the commonly applied blue-shifted BRET2 system, consisting of the Renilla luciferase Rluc2 and the large-stokes shift fluorescent protein GFP2, suffers from light absorption and light scattering in human plasma samples. To address this challenge, we developed a red-shifted BRET-based plasmin sensor by substituting BRET2 with the BRET6 system. BRET6 is composed of the red-shifted RLuc8.6 luciferase linked to the red light emitting fluorescent protein TurboFP635. The BRET6 biosensor exhibited 3-fold less light absorption in plasma samples compared to the BRET2 sensor leading to an up to a 5-fold increase in sensitivity for plasmin detection in plasma. The limits of detection for plasmin were determined to be 11.90 nM in 7.5% (v/v) plasma with a 10 min assay which enables biologically relevant plasmin activities of thrombolytic therapies to be detected. While a colorigenic plasmin activity assay achieved a similar detection limit of 10.91 nM in 7.5% (v/v) human plasma, it required a 2 h incubation period. The BRET6 sensor described here is faster and more specific than the colorigenic assay as it did not respond to unspiked human plasma samples. |
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Proteases are key signalling molecules for many physiological processes and their dysregulation is implicated in the progression of a range of diseases. Sensitive methods to measure protease activities in complex biological samples are critical for rapid disease diagnoses. The proteolytic activity of plasmin reflects the fibrinolysis state of blood and its deregulation can indicate pathologies such as bleeding events. While Bioluminescence Resonance Energy Transfer (BRET) is a powerful and sensitive method for the detection of protease activity, the commonly applied blue-shifted BRET2 system, consisting of the Renilla luciferase Rluc2 and the large-stokes shift fluorescent protein GFP2, suffers from light absorption and light scattering in human plasma samples. To address this challenge, we developed a red-shifted BRET-based plasmin sensor by substituting BRET2 with the BRET6 system. BRET6 is composed of the red-shifted RLuc8.6 luciferase linked to the red light emitting fluorescent protein TurboFP635. The BRET6 biosensor exhibited 3-fold less light absorption in plasma samples compared to the BRET2 sensor leading to an up to a 5-fold increase in sensitivity for plasmin detection in plasma. The limits of detection for plasmin were determined to be 11.90 nM in 7.5% (v/v) plasma with a 10 min assay which enables biologically relevant plasmin activities of thrombolytic therapies to be detected. While a colorigenic plasmin activity assay achieved a similar detection limit of 10.91 nM in 7.5% (v/v) human plasma, it required a 2 h incubation period. The BRET6 sensor described here is faster and more specific than the colorigenic assay as it did not respond to unspiked human plasma samples. |
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