A Multidisciplinary Approach to Protein Nanoarrays
Lead Research Organisation:
University of Manchester
Department Name: Chemistry
Abstract
Proteins are the molecular machinery of all living organisms and perform all the functions necessary for life. In every organism, large numbers of proteins act in a highly orchestrated manner to perform tasks from the processing of nutrients to the reproduction of the organism. The function of proteins therefore has a major bearing on health as many diseases are caused by the altered activity, deficiency or overproduction of various proteins. The activity of proteins also underlies any human economic activity which is reliant on living systems such as industries which utilise fermentation and the agricultural sector. Thus, in order to fully understand living systems, there is a need for the identification of proteins, measurement of the amounts present, discovery of their function and elucidation of the mechanisms by which they interact with each other. These are encompassed in the scientific field known as proteomics . One method of enabling the study of proteins is to anchor them to a two-dimensional surface, such as a glass slide or chip , where each protein is placed at a defined location on the surface. This offers a convenient means of handling large numbers of proteins and a means to test them simultaneously. In this way, an entire chip and it's collection of proteins can be subjected to various tests and if a biological activity of interest is detected at a particular location on that slide, the protein which caused that activity can be identified. Current technology allows the production of arrays of approximately 10,000 protein spots on a single chip with spot sizes of about a hundredth of a millimetre.However, the number of proteins in nature that could be examined is vast. In humans alone, the Human Genome Project has identified approximately 50,000 proteins. Moreover, the types and activity of various proteins are variable between different cells and at different times in a cell's life cycle. Many interesting proteins are also present in very small amounts. To be able to examine such a large number of proteins from such widely varied sources, production methods are needed which further increase the number of proteins that can be placed on a chip for analysis. Chips with smaller protein spots would also mean that only tiny amounts of proteins which may be rare are needed for testing. Further miniaturisation these spots could be achieved by harnessing the techniques developed in nanotechnology, the science of constructing objects at nanometre scales (a billionth of a metre) and in principle, down to even a single molecule. Accordingly, this proposal aims to use two nanotechnological techniques to construct these protein arrays on siloxane surfaces, a glass-like material. These techniques are dip-pen nanolithography, where a very fine (nanometre wide) tip is dipped in a chemical ink and used to write patterns on surfaces, and scanning near-field photolithography which uses a very fine hole to direct laser light to write patterns on the surface. However, this proposal also includes a number of other scientific areas which will be needed to build a protein nanoarray . Molecular biology techniques will be employed to produce proteins which can be specifically attached to areas on the surface that have been patterned such that the way in which the protein is attached is well defined. To bring the surface nanotechnology and biology together, synthetic chemistry will be employed to prepare novel inks which are compatible with biological systems, suitable for writing and can react under laser light to produce spots (or other patterns) which can subsequently attach proteins in a specific manner. While there have already been examples where nanotechnology has been used to make arrays of this scale with one or two proteins, the key breakthrough that is being proposed here is an array of multiple proteins which would be directly relevant in proteomics.
Publications
Wong L. S.
(2009)
Site-selective covalent protein immobilisation on nanofabricated surfaces mediated by a phosphopantetheinyl transferase towards nanomedical arrays and biosensors
in JOURNAL OF PHARMACY AND PHARMACOLOGY
Wong LS
(2009)
Selective covalent protein immobilization: strategies and applications.
in Chemical reviews
Wong Lu Shin
(2009)
Application of phosphopantetheinyl transferase catalyzed site-selective covalent protein immobilization on to nanofabricated surfaces
in ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY
Wong LS
(2010)
Nanoscale biomolecular structures on self-assembled monolayers generated from modular pegylated disulfides.
in Chemistry (Weinheim an der Bergstrasse, Germany)
Wong LS
(2010)
Site-selective immobilisation of functional enzymes on to polystyrene nanoparticles.
in Organic & biomolecular chemistry
Ul Haq E
(2010)
Parallel scanning near-field photolithography: the snomipede.
in Nano letters
Ul-Haq E
(2011)
The Snomipede: A parallel platform for scanning near-field photolithography
in Journal of Materials Research
Chai J
(2011)
Single-molecule protein arrays enabled by scanning probe block copolymer lithography.
in Proceedings of the National Academy of Sciences of the United States of America
Ahmad SA
(2011)
Protein micro- and nanopatterning using aminosilanes with protein-resistant photolabile protecting groups.
in Journal of the American Chemical Society
Description | This research investigated methods to place proteins on to surfaces with nanometre resolution, and to study the behaviour of the proteins when confined at this scale. In the process, methods for the placement of single protein molecules were developed, and it was found that surface features containing proteins that were smaller resulted in apparently higher protein activity. |
Exploitation Route | The findings in this project have initiated new research projects in a range of avenues, as evidenced by further grant awards. |
Sectors | Aerospace, Defence and Marine,Pharmaceuticals and Medical Biotechnology,Other |
Description | BBSRC Grouped |
Amount | £472,039 (GBP) |
Funding ID | BB/H011080/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 10/2011 |
End | 10/2014 |
Description | BBSRC Grouped |
Amount | £472,039 (GBP) |
Funding ID | BB/H011080/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 10/2011 |
End | 10/2014 |
Description | British Council Newton Fund Institutional Links |
Amount | £149,933 (GBP) |
Funding ID | 216196834 |
Organisation | British Council |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 09/2016 |
End | 09/2018 |
Description | Metals in Biology: The elements of Biotechnology and Bioenergy |
Amount | £325,410 (GBP) |
Funding ID | BB/L013711/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 01/2014 |
End | 01/2018 |
Description | MultiUSer equipment for high-throughput, high-content analysis in Industrial and Cellular biotechnology (MUSIC) |
Amount | £277,784 (GBP) |
Funding ID | BB/R000093/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 08/2017 |
End | 08/2018 |
Description | Royal Pharmaceutical Society of Great Br |
Amount | £250 (GBP) |
Organisation | Royal Pharmaceutical Society of Great Britain |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 09/2009 |
End | 09/2009 |
Title | LED-based Photochemistry Equipment |
Description | Development of small-scale photochemistry equipment using narrow wavelength LED light sources. |
Type Of Material | Improvements to research infrastructure |
Year Produced | 2016 |
Provided To Others? | Yes |
Impact | EPSRC Fellowship awarded to Dr. Daniele Leonori |
Description | Liverpool John Moores (Dr. C. R. Coxon) - Bioorganic chemistry |
Organisation | Liverpool John Moores University |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Co-supervision of PhD student at LJMU, access to photochemistry equipment at Manchester |
Collaborator Contribution | Provision of PhD student, peptide synthesis facilities |
Impact | Training of PhD student. |
Start Year | 2016 |
Description | Universiti Putra Malaysia (Dr. S. A. Alang Ahmad) |
Organisation | Putra Malaysia University |
Department | Institute of Biosciences |
Country | Malaysia |
Sector | Academic/University |
PI Contribution | Nanofabrication of DNA arrays and surface analysis of arrays. |
Collaborator Contribution | Production of DNA-nanoparticle conjugates and testing of diagnostic assays. |
Impact | Delivered two seminars at UPM in 2012 and 2016. |
Start Year | 2016 |
Title | Algorithm for automated probe array alignment for use with scanning probe nanolithography |
Description | The precision and versatility afforded by scanning probe microscopy has enabled the development of a variety of methods for the facile fabrication of user-defined patterns on a variety of surfaces with nanoscale resolution. Historically, the major limitation of such scanning-probe nanolithography has been the inherently low throughput of single probe instrumentation, which has been addressed by the use of "two-dimensional" arrays of multiple probes for parallelised nanolithography. Key to the successful implementation of such arrays is a means to accurately align them relative to the substrate surface, such that all probes come into contact with the surface simultaneously upon the commencement of lithography. Here, an algorithm for the rapid, accurate and automated alignment of an array is described in the context of polymer pen lithography. This automation enables the alignment of the array of probes within minutes, without user intervention. Subsequent nanolithography of thiols on gold substrates demonstrated the generation of features over large (cm2) areas with high uniformity. Example features were 66.5 ± 9.8 and 71.3 ± 9.3 nm in size across a distance of 1.4 cm, indicating any misalignment as =0.0003°. |
IP Reference | |
Protection | Copyrighted (e.g. software) |
Year Protection Granted | 2016 |
Licensed | Commercial In Confidence |
Impact | Algorithm used by researchers using the scanning probe nanolithography equipment at the University of Manchester |
Title | GENERATION OF COMBINATORIAL PATTERNS BY DELIBERATE TILTING OF A POLYMER-PEN ARRAY |
Description | The disclosure relates to a method of forming a pattern having pattern elements with a plurality of sizes on a substrate surface with a tilted pen array that includes choosing a tilt geometry for a pen array with respect to a substrate, inducing the tilt geometry between the pen array and the substrate surface, and forming a pattern having pattern elements on the substrate surface with the titled pen array, whereby the size of the formed pattern elements varies across the substrate surface along the tilted axis or axes. For example, the tilt geometry is in reference to the substrate surface and comprises a first angle with respect to a first axis of the substrate and a second angle with respect to a second axis of the substrate, the second axis being perpendicular to the first axis, and at least one of the first and second angles being non-zero. |
IP Reference | WO2011071753 |
Protection | Patent granted |
Year Protection Granted | 2011 |
Licensed | Commercial In Confidence |
Impact | n/a |
Title | SUBSTRATES HAVING NANOSTRUCTURES HAVING BIOLOGICAL SPECIES IMMOBILIZED THEREON AND METHODS OF FORMING THE SAME AND METHODS OF FORMING NANOSTRUCTURES ON SURFACES |
Description | A method for controlling a number of biological species immobilized on a nanostructure can include selecting a number of biological species to be immobilized on a nanostructure, the biological species having a hydrodynamic diameter, patterning a nanostructure using scanning probe block copolymer lithography, the patterned feature being formed to have a diameter (or line width) to form a nanostructure having a diameter (or line width) corresponding to (a) the hydrodynamic diameter of the biological species and (b) the number of biological species selected to be immobilized on the nanostructure, and immobilizing the biological species on the nanostructure, wherein the selected number of biological species are immobilized on the nanostructure |
IP Reference | WO2013049409 |
Protection | Patent granted |
Year Protection Granted | 2013 |
Licensed | Commercial In Confidence |
Impact | n/a |
Description | Interview by RCUK |
Form Of Engagement Activity | A press release, press conference or response to a media enquiry/interview |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | Interview by RCUK as a case study to be posted on their website. Intended to demonstrate the career paths of researchers to the general public. |
Year(s) Of Engagement Activity | 2012 |
URL | http://www.rcuk.ac.uk/skills/percase/Chem/Wong/ |