Nanowells and Nanocages for High Throughput Protein Recognition

Lead Research Organisation: Imperial College London
Department Name: Dept of Chemistry

Abstract

Biosensors are an integral and increasingly important part of modern biomedicine and new developments in biosensor technologies are increasingly seeing their application in the process industry, security, environmental and biodefense application markets. We seek to address the limitations of current anti-body based diagnostics and we propose the development of a radical new nanotechnology for high throughput electronic recognition of proteins. While there are some moves to develop nanotechnology approaches to biosensing, these have yet to make a mark and there remains an unmet need in the development of lab-on-chip biosensors that are affordable, integrated, fast, capable of multiplexed detection and monitoring, and highly sensitive which can detect trace levels of analyte. This proposal directly addresses these issues.

NB this summary is necessarily brief to avoid potential issues of disclosure.

Technical Summary

Biosensors are an integral and increasingly important part of modern biomedicine and new developments in biosensor technologies are increasingly seeing their application in the process industry, security, environmental and biodefense application markets. We seek to address the limitations of current anti-body based diagnostics and we propose the development of a radical new nanotechnology for high throughput electronic recognition of proteins. While there are some moves to develop nanotechnology approaches to biosensing, these have yet to make a mark and there remains an unmet need in the development of lab-on-chip biosensors that are affordable, integrated, fast, capable of multiplexed detection and monitoring, and highly sensitive which can detect trace levels of analyte. This proposal directly addresses these issues.

NB this summary is necessarily brief to avoid potential issues of disclosure.

Planned Impact

This project will deliver an unprecedented advance in single-molecule, label-free sensing that will contribute substantially to the UK's global leadership in this area. The major outputs will be (i) a step-change in the development of new analytical and biophysical tools and (ii) interdisciplinary training for early-career researchers. (iii) This proposal also contributes to the current BBSRC Strategic Research Priority of Technology development for the biosciences. We anticipate that this will benefit policymakers, funding bodies and academic institutions by providing clear evidence of the value of interdisciplinary research for the future of UK science.

This proposal will lead to direct impact in a number of different areas:

Communication and engagement: this will involve a number of third parties including industrial partners, government agencies such as TSB and RCUK; public engagement via ongoing activities through schools and social media; communication through dissemination activities involving publications and talks.

Academic Collaborations: the applicants on this grant all have numerous academic collaborators who can both provide input into and benefit from this research. Additionally the radical nature of this research proposal provides an opportunity to seek new collaborations and industrial partners.

Industrial Collaborations: the radical nature of this research proposal provides new opportunities to develop both existing and potentially new collaborations with industrial partners.

Exploitation: the innovative nature of this proposal means that it will likely lead to new IP that will be secured and exploited.

Transferable skills: the training provided to the researcher, on this project will increase the availability of highly skilled workers in the UK that will be an advantage in a knowledge-based economy. In addition it will also lead to enhanced skills and knowledge for the leading academics that will inform their ability to progress the future development of this project.

Outreach: all of the academics in this proposal engage in various types of outreach activities to inform, inspire and educate.

Publications

10 25 50
 
Description Through this work several properties of a new bacterio-ferritin construct have been examined.

Storing the cell pellets and purifying as required proved to be successful. The cell pellets could be stored at -80 °C and used for a maximum of 5 weeks.

The stability study on ZZ-GFP-Bfr and Bfr-Au nanocage placed the maximum storage at 4 weeks at -20 °C. The ob-served fragmentation was likely to occur from chemical deg-radation32 enabled by the tertiary structure of the folded pro-tein. Thus further work is required to engineer a more stable form of the protein and work out exactly how ZZ-GFP-Bfr is fragmenting.

Citrate stabilized nanoparticles appeared to dissociate less rapidly than the original nanoparticles. However as only one study was conducted further work is needed to confirm this observation.

The gold binding domain in this construct was added to prevent the spontaneous formation of Bfr-Au nanocage and drive the equilibrium towards Bfr-Au nanocage once gold nanoparticles were added. Whilst the studies in this re-search have proved this has not been successful in driving the equilibrium towards the 24-mer form, the observations have been supported by literature. A possible solution to this problem could be through adding CaCl2 to the mix. As well as probing other constructs.

Polydispersity was also observed during ZZ-GFP-Bfr puri-fication on the FPLC, due to multimers, commonly found in literature7. There was also some evidence of spontaneous nanocage formation without the presence of gold. In future work native PAGE will be used alongside these other tech-niques to monitor the polydispersity of solutions.

No endpoint was reached when studying Bfr-Au nanocage formation. Hence the amount of gold added was the limiting factor. Whilst there was a limit to this experiment, in future work nanoparticle sizing will be used to track formation on a smaller scale.

When studying the amount of gold added, a maximum was achieved under stoichiometric conditions and this will be used in future experimentation.

Due to the high salt concentration and increased amount of gold for stoichiometric addition aggregation occurred. A microplate study on aggregation during Bfr-Au nanocage formation using citrate nanoparticles showed aggregation in both a low and high salt buffer. Thus the gold nanoparti-cles used in this experimentation need to be studied further. A separate look into ways to combat the aggregation of the gold nanoparticles would be invaluable. This could be through changing the buffers used or perhaps using differ-ently stabilised nanoparticles.
Exploitation Route This work has provided an interesting insight into a new protein construct ZZ-GFP-Bfr. Many of the observations have directly correlated to literature and helped further build the complex picture of engineered protein nanocages. Whilst much is to be done before ZZ-GFP-Bfr is suitable for use in single molecule detection experiments, this construct has enabled further study into the intricate nanocage for-mation process with bacterioferritin. Supporting much pre-vious work on native ferritin and native/engineered bacteri-oferritin. The possibility of chemical degradation causing the fragmentation of the construct is a valuable find and with more study could enable further understanding of pro-tein chemical degradation. This work has also sufficed to highlight some of the problems associated with gold nano-particle aggregation.
Sectors Chemicals,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description ERC Consolidator Grant
Amount € 1,970,000 (EUR)
Organisation European Research Council (ERC) 
Sector Public
Country European Union (EU)
Start 09/2017 
End 09/2022
 
Description ERC Proof of Concept Grant
Amount € 150,000 (EUR)
Organisation European Research Council (ERC) 
Sector Public
Country European Union (EU)
Start 01/2016 
End 06/2017
 
Description ICB studentship
Amount £80,000 (GBP)
Organisation Imperial College London 
Department EPSRC Centres for Doctoral Training
Sector Academic/University
Country United Kingdom
Start 10/2014 
End 09/2018
 
Title new sensing strategies 
Description As part of this grant new sensing strategies were developed which can be used for confirming stability of proteins 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact patent application submitted and high impact paper published. Currently looking into commercialisation. 
 
Title DEP Trapping Using Metalized Nanopipettes 
Description Nanopore sensing using DEP traps 
IP Reference P58630GB 
Protection Patent application published
Year Protection Granted 2015
Licensed No
Impact NA
 
Description 2nd London Chemical Biology Colloquium, June 30th, 2014 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation keynote/invited speaker
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact shared ideas on nano pore sensing for detection of biological molecules

invited to give further lectures at international institutions
Year(s) Of Engagement Activity 2014
 
Description Imperial Outreach - RSC Chemistry Day, October 22nd, 2014 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Outreach activity with high school students

Students introduced to subject matter of my research
Year(s) Of Engagement Activity 2014
 
Description Medical Nanospectroscopy conference UK 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A talk presented on Real-Time Dopamine Sensing
Year(s) Of Engagement Activity 2015
 
Description Micro and Nano Flows, September7-10, 2014 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation keynote/invited speaker
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact introduced concepts on nano pore sensing

described potential uses for nano pore sensing which resulted on colleagues coming up with new applications.
Year(s) Of Engagement Activity 2014
 
Description microfluidics world congress london 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Novel Strategies in Single Molecule Sensing
Year(s) Of Engagement Activity 2015