HLT04-BioSurf

Deliverable
number

Deliverable description

1.1.1

Requirements document outlining the requirements for all activities in WP1-4

1.1.2

Two protocols for the production of planar standard platforms on silicon oxides and gold

1.1.3

Acquisition of the appropriate carbohydrate probes

1.1.4

Acquisition of the appropriate peptide and protein probes

1.1.5

Protocol for preparation of a planar carbohydrate reference surface, and a carbohydrate reference array

1.1.6

Production of planar carbohydrate reference surface samples and arrays, as per the requirements document for JRP HLT04

1.1.7

Protocol for the preparation of at least two planar protein reference surfaces

1.1.8

Protocol for the preparation of at least four planar peptide reference surfaces

1.1.9

Production of planar peptide and protein reference surface samples, as per the requirements document for JRP HLT04

1.1.10

Uncertainty budget for measuring probe density on planar surfaces

1.2.1

XPS data of the investigative phase of Task 1.1

1.2.2

XPS data of platforms and surfaces produced in Task 1.1

1.2.3

QCM and ellipsometry data during linker and passivator attachment to substrates and probe attachment to platforms

1.2.4

ToF-SIMS and infrared data from platforms and surfaces

1.2.5

Three analytical protocols (QCM, ellipsometry, ToF-SIMS), to measure the density of linker and probe molecules

1.2.6

Uncertainty budgets and traceability chains for the analytical approaches which demonstrate repeatability to better than 10%

1.2.7

Good practice guide describing protocols, comparability of methods and uncertainties for QCM, ellipsometry, ToF-SIMS

1.3.1

Production of reference NP surface samples

1.3.2

Data sets from XPS, DLS, DCS and zeta potential for NP substrates, standard NP platforms and reference NP surfaces

1.3.3

Data sets from SAXS for NP substrates, standard NP platforms and reference NP surfaces

1.3.4

Protocol on traceable size determination for NP substrates, standard NP platforms and reference NP surfaces using SAXS

1.3.5

Protocol on surface chemistry determination using XPS for NP substrates, standard NP platforms and reference NP surfaces

1.3.6

Protocol on size and density determination protocols using DLS and DCS for NP substrates, standard NP platforms and reference NP surfaces

1.3.7

Report on the ability of zeta potential measurements to provide information on the number of attached probes per NP

1.4.1

Protocol outlining the quality control of the production of reference carbohydrate microarray, protein and peptide surfaces

1.4.2

Protocol outlining the quality control of the production of reference NPs

1.4.3

Validation of quality control methods for planar reference surfaces by XPS and SIMS

1.4.4

Report detailing the quality control methods for planar reference surfaces by XPS and SIMS, optical methods (fluorescence, IR spectroscopy) and water contact angle measurements. 

1.4.5

Recommendations, to ensure a reasonable shelf life, will be made

1.5.1

Submission of the inter-laboratory comparison as a VAMAS project

1.5.2

Confirmation of at least 10 participants for the inter-lab comparison

1.5.3

Flow chart showing the organisation of the inter-lab comparison

1.5.4

Production of sufficient planar reference surfaces for all participating laboratories

1.5.5

Confirmation that all reference samples have been delivered to the participants in the inter-lab comparison

Deliverable number

Deliverable description

2.1.1

The dependence of spectral intensities in GCIB-SIMS to surface concentration using planar peptide reference surfaces developed in task 1.1

2.1.2

Measurements of the sensitivity of SIMS to effects such as molecular size, attachment method, orientation and denaturation

2.1.3

Measurements of the ability of GCIB-SIMS to distinguish structurally similar molecules at surfaces

2.1.4

Recommendations for data analysis and the optimum size and energy of the cluster ion

2.1.5

Measurements of the sensitivity of GCIB-SIMS to changes in probe structure

2.1.6

A method for determining the identity of the probe structure using GCIB-SIMS

2.1.7

Data from the application of the methods developed for planar peptide reference surfaces to planar protein reference surfaces and planar carbohydrate reference surfaces

2.1.8

An analytical procedure describing the methods for data interpretation for GCIB-SIMS

2.2.1 (REG(TUB) D1.5)

Final design of the UHV compatible liquid cells

2.2.2

Confirmation of the  biomolecular sample systems to be investigated

2.2.3 (REG(TUB) D1.6)

Production of at least 10 UHV compatible liquid cells 

2.2.4 (REG(TUB) D1.7)

Report on the testing of the UHV compatible liquid cells

2.2.5

Protocol for generating the reference surfaces, from task 1.1, with at least three different probe densities

2.2.6

Data verifying that the probes have been reproduced on at least three different probe densities on the windows of the UHV compatible liquid cells by XPS

2.2.7

Protocol describing the method for optimising the angular and energetic range of GIXRF-NEXAFS for biomolecules  attached at the Si/SiO2 interface of the UHV compatible liquid cell windows

2.2.8 (REG (TUB) D1.9)

Data from GIXRF-NEXAFS experiments in the soft x-ray range, to derive the chemical binding state of biomolecules deposited at the Si/SiO2 interface of the thin Si3N4 membrane of the UHV compatible liquid cells

Deliverable number

Deliverable description

3.1.1

Confirmation of the bound targets chosen for task 3.1

3.1.2

Measurements of the amount of bound target using in-situ ellipsometry and QCM-D

3.1.3

Estimation of the binding affinities and dissociation rates between target and reference surface

3.1.4

Estimation of the dynamic measurements equilibrium and rate constants for the binding

3.1.5

Repeatability and uncertainty budget for the amount of bound target will be established

3.2.2

Confirmation of the selection of labelled biomolecule for task 3.2

3.2.3

At least one labelled biomolecule in sufficient quantities for molecular orientation experiments using XRF/NEXAFS

3.2.6

X-ray spectrometric investigations in a UHV-compatible liquid cell for different stages of target binding to the diagnostic Si interface of the membrane.

3.3.1

Confirmation of the choice of designated targets and one non-target to be used in task 3.3

3.3.2

Size measurements using DLS and DCS of the reference nanoparticle surfaces from D1.3.1.

3.3.3

Report on the assessment of the possibility of measuring target binding through changes in optical absorption or zeta potential

3.3.4

Measurements of target binding on the reference nanoparticle surfaces from D1.3.1.

3.3.5

Diameter of core-shell nanoparticles after attachment of the target determined using SAXS

3.3.6

Uncertainty analysis on the SAXS method from D3.3.5, and determination of the limit of detection.

3.3.7

Report on methods for determining the diameter of the reference NP surfaces in following the attachment of targets.

3.4.1

Software plan of the physical model to be implemented in the numerical code for the biomolecular simulation software, focusing on PB and MD models

3.4.2

Numerical analysis software based on PB equation and its integration into a MD numerical model for the simulation of the binding mechanism

3.4.3

Report on the comparison between measured and experimental results on binding affinity as a function of probe type

3.4.4

Report on the comparison between measured and experimental results on binding affinity as a function of probe orientation

3.4.5

Design rules, suitable for end-users, on the definition of the optimal density and orientation of probes at a surface in order to obtain the most reproducible diagnostic response

Deliverable number

Deliverable description

4.1.1 REG(Chalmers) D1.5

New prototype instrument based on waveguide optics and TIRF-microscopy for single target detection using liposome-conjugated probes.

4.1.2 REG(Chalmers) D2.1

Confirmation of the selection of a suitable probe-target pair for task 4.1

4.1.3 REG(Chalmers) D2.2

Procedure for the conjugation of the antibodies to the liposome and surface

4.1.4 REG(Chalmers) D2.3

Investigation into the effect of varying probe concentration on the surface and in solution using the probe-target pair for task 4.1

4.1.5 REG(Chalmers) D2.4

Evaluation of the sensitivity, specificity and quantification capability of the waveguide-based method to detect the target

4.1.6 REG(Chalmers) D2.5

Procedure for the optimisation of existing TIRF-microscopy based assays designed for single target detection using liposome-conjugated probes

4.1.7 REG(Chalmers) D2.6

Confirmation of the choice of samples to be used to evaluate the ability of the liposome-based TIRF method for detecting multiplexed proteins in a selected real biological sample (e.g. blood serum)

4.1.8 REG(Chalmers) D2.7

Evaluation of the sensitivity, specificity and quantification capability of the method to detect amyloid-β peptides in complex samples using the TIRF assay

4.1.9 REG(Chalmers) D2.8

Multiplexed protein detection using TIRF and liposomes with different organic dyes

4.1.10 REG(Chalmers) D2.9

Report on the evaluation of the all-polymer waveguide-based assay and comparison with TIRF using the chosen amyloid-β peptides and the probe density of a biorecognition substrate

4.2.1

Procedure for an imaging MS based method for single target detection using liposome-conjugated antibodies

4.2.2

Protocol for the verification of preparation steps of the imaging MS based method using QCM-D and fluorescence microscopy

4.2.3 REG(Chalmers) D3.1

Confirmation of the selected suitable probe-target pair for task 4.2

4.2.4

Procedure for conjugation of the selected probes to the liposome and surface

4.2.5 REG(Chalmers) D3.2

Detection of surface-immobilised liposomes using the developed imaging TOF-SIMS based method

4.2.6

Investigation into the effect of varying probe concentration on the surface and in solution using the probe-target pair for task 4.2

4.2.7

Report on the evaluation of the developed imaging TOF-SIMS based method with respect to sensitivity, specificity and quantification capability for a single target

4.2.11 REG(Chalmers) D3.5

Confirmation of the samples selected to evaluate the ability of the imaging TOF-SIMS based method for detecting multiplexed proteins in a selected real biological samples (e.g. blood serum)

4.3.1

Procedure for ambient MS methods for the detection and identification of single probes and targets on planar reference surfaces

4.3.2

Report on the evaluation of the ambient MS based detection methods with respect to their sensitivity, specificity and reliability

4.3.3

Report on the effect of molecular weight and protein composition on the detection efficiency of the ambient MS based detection methods

4.3.4

Optimised procedure for ambient MS methods including recommendations on the applicability of the methods for diagnostic application

4.3.5

Confirmation of the selected targets and probes to be used for ambient MS based methods to image reference arrays

4.3.6

Measurements using ambient MS based methods to image reference arrays, and identify the targets bound to different probe spots.

4.3.7

Procedure for quantifying the amount of bound target on the reference arrays using the ambient MS based methods