Final Report Summary - PROTEPROBE (Electrically Controlled Protein Conformation on 3D Tissue Scaffolds)
ProtEprobe was aimed at developing a new technique to probe and sense intricate molecular activity within complex biological environments. High sensitivity factor silver nanoplate biosensors, highlighted as a potentially excellent tool for in situ monitoring of protein folding and conformation have been exploited to optically record protein conformational changes. Electrical recording was accomplished using organic electrochemical transistors (OECTs). The fellow has successfully demonstrated a novel system for molecular detection in celluar environments through poineering fusion of these two cutting edge technologies. This project focused on first progressing the silver nanoplate technology to optically sense protein conformational changes within cellular environments and subsequently establishing novel OECT recording of nanoplates. Finally OECT recording of biomolecules using biofunctionalised nanoplates was demonstrated in a system which can provide synergistic dual optical and electrical high sensitive detection. Importantly this new technique adds specificity capability to OECT measurements.
Researching and developing such moleuclar probes required the fellow to combine expertise in nanotechnology and plasmonics with newly acquired knowledge gained during the course of the project to date in the areas of bioelectronics including OECT operation, cell culture and protein conformational behaviour. The fellow trained on the physics of organic electronic devices, cell culture techniques including fluorescent labelling and microscopy and in vitro control of protein conformation via pH and salinity and ionic modulation. Following insight and knowledge gained by the fellow during training the fellow has taken the ambitious approach to carry out the first coordination of OECT electrical measurements of biofunctionalised nanoplates for the provision of bio-specificity to OECT measurements.
The fellow designed and operated a proof-of-principle prototype for high sensitive biomoleuclar detection by combining the fellows nanoplate biosensing technology and the hosts OECT platform.
Versatile optical protein conformational monitoring in cellular environments
The fellow has succeeded in implementing gold edge-coated triangular silver nanoplates to monitor conformational transitions of the ubiquitous cellular protein fibronectin (Fn), both in solution phase and for the first time within living cells. A straightforward no-wash assay for the serum detection of Fn is demonstrated which can readily be optimised with potential for use within the clinical settings for the disease assessment such as cancer metastasis has also been demonstrated. The progression of Fn diffusely bound to cells in a compact state to form Fn fibril matrices in which Fn displays a highly extended conformation generated very large nanoplate spectral shifts of 156 nm over 48 hours correlating with Fn models in which Fn continuously unfolds and extends within cells, binding to form fibrils which grow and become thicker in an evolving extra cellular matrix. The versatility and label-free feature of the nanoplates and in particular their capability to detect and monitor protein structural and conformational transitions in cellular environments presents them as a powerful new tool to signature protein conformational activity in living cells.
OECT measured biofunctionalised nanoplates
In the case of the OECTs the ionic nature of the nanoplate impact on the resistance and capacitance of the system. This work has shown that nanoplates, both biofunctionalised and unfunctionalised show highly reproducible impact on the transconductance cut off frequency of the OECTs. A characteristic shift in the transconductance has been demonstrated for the two different conformations of Fn, (extended and compact) demonstrating the capacity of the nanoplate OECT systems to clearly differentiate between two different protein conformational states.
In an important step the nanoplate OECT system was demonstrated for the detection of the small cytokine molecule, tumour necrosis factor alpha (TNFα), which is particularly prevalent for brain function and in the onset of brain seizures. Using nanoplates functionalised with two different antibodies specific to TNFα and also nanoplates functionalised with an antibody not specific TNFα as a control, modulation of OECT transconductance frequency in specific response to TNFα was clearly demonstrated. These measurements were further extended to in serum detection where specific detection of TNFα at levels as low as 6pg/ml were recorded.
The fellow's successful fusion of these two technologies, summarised in table I, has achieved a seried of advances and benifits beyond the state of the art. In particular the addition of specificity and biofunctionalisation capability to OECT measurements which is identified as an important limitation of OECTs has been achieved through the features of biofunctionaised nanoplates.
Researching and developing such moleuclar probes required the fellow to combine expertise in nanotechnology and plasmonics with newly acquired knowledge gained during the course of the project to date in the areas of bioelectronics including OECT operation, cell culture and protein conformational behaviour. The fellow trained on the physics of organic electronic devices, cell culture techniques including fluorescent labelling and microscopy and in vitro control of protein conformation via pH and salinity and ionic modulation. Following insight and knowledge gained by the fellow during training the fellow has taken the ambitious approach to carry out the first coordination of OECT electrical measurements of biofunctionalised nanoplates for the provision of bio-specificity to OECT measurements.
The fellow designed and operated a proof-of-principle prototype for high sensitive biomoleuclar detection by combining the fellows nanoplate biosensing technology and the hosts OECT platform.
Versatile optical protein conformational monitoring in cellular environments
The fellow has succeeded in implementing gold edge-coated triangular silver nanoplates to monitor conformational transitions of the ubiquitous cellular protein fibronectin (Fn), both in solution phase and for the first time within living cells. A straightforward no-wash assay for the serum detection of Fn is demonstrated which can readily be optimised with potential for use within the clinical settings for the disease assessment such as cancer metastasis has also been demonstrated. The progression of Fn diffusely bound to cells in a compact state to form Fn fibril matrices in which Fn displays a highly extended conformation generated very large nanoplate spectral shifts of 156 nm over 48 hours correlating with Fn models in which Fn continuously unfolds and extends within cells, binding to form fibrils which grow and become thicker in an evolving extra cellular matrix. The versatility and label-free feature of the nanoplates and in particular their capability to detect and monitor protein structural and conformational transitions in cellular environments presents them as a powerful new tool to signature protein conformational activity in living cells.
OECT measured biofunctionalised nanoplates
In the case of the OECTs the ionic nature of the nanoplate impact on the resistance and capacitance of the system. This work has shown that nanoplates, both biofunctionalised and unfunctionalised show highly reproducible impact on the transconductance cut off frequency of the OECTs. A characteristic shift in the transconductance has been demonstrated for the two different conformations of Fn, (extended and compact) demonstrating the capacity of the nanoplate OECT systems to clearly differentiate between two different protein conformational states.
In an important step the nanoplate OECT system was demonstrated for the detection of the small cytokine molecule, tumour necrosis factor alpha (TNFα), which is particularly prevalent for brain function and in the onset of brain seizures. Using nanoplates functionalised with two different antibodies specific to TNFα and also nanoplates functionalised with an antibody not specific TNFα as a control, modulation of OECT transconductance frequency in specific response to TNFα was clearly demonstrated. These measurements were further extended to in serum detection where specific detection of TNFα at levels as low as 6pg/ml were recorded.
The fellow's successful fusion of these two technologies, summarised in table I, has achieved a seried of advances and benifits beyond the state of the art. In particular the addition of specificity and biofunctionalisation capability to OECT measurements which is identified as an important limitation of OECTs has been achieved through the features of biofunctionaised nanoplates.