Final Report Summary - CE-MICROARRAY (Cavity Enhanced Microarray as an Ultra-sensitive Tool to aid Sepsis Diagnosis)
Executive Summary:
The CE-microArray project was a two-year programme (extended by a total of eight months via a five-month suspension and three-month extension period) which had two main aims. Firstly, to increase the sensitivity of detection of colorimetric ELISAs and microarrays by developing novel microtiter plates and plate readers based on cavity enhanced absorption spectroscopy. The secondary aim was to develop a novel bioassay which could be used to improve the sensitivity and selectivity of sepsis risk diagnosis. Sepsis is a life-threatening illness caused by the body’s overreaction to an infection and can be triggered either directly by infection or may occur after medical treatment or surgery. The mortality rate in patients admitted to hospital with severe sepsis is 28-50% and it remains the most prevalent cause of death in non-coronary Intensive Care Units (ICUs ).
The CE-microArray project led to the development of three types of cavity enhanced devices. Firstly, a cavity enhanced microtiter plate which had high reflectivity dielectric mirrors deposited on the underside of the plate and on the coverplate. This led to a self contained cavity enhanced device which was able to provide a five fold increase in the path length and sensitivity of measurement compared to a conventional microtiter plate. The second device was a cavity enhanced microplate reader which was able to make single point measurements in the wells of a standard 96 well microtiter plate with greater than 30 fold increase in sensitivity when compared to measurements with a conventional microtiter plate reader. The final device was a cavity enhanced imaging microtiter plate reader was capable of making imaging measurements on colorimetric microarrays with a 6-8 fold increase in sensitivity.
In parallel to the development of the cavity enhanced devices, consortium partners developed a novel bioassay for sepsis risk diagnosis based on choosing a selection of biomarkers identified by experts in the field and then developing suitable assays for the biomarkers. Calibration samples were also provided to the test the relative sensitivity of the cavity enhanced devices compared to conventional plate readers.
Project Context and Objectives:
The overall objectives of the CE-microArray project were the production of cavity enhanced microtiter plates (MTP) and cavity enhanced microplate readers which will result in up to a 100 fold increase in the sensitivity of colorimetric bioassays, leading to higher specificity for target analytes as well as cost reductions from the use of smaller quantities of biochemicals. In addition a novel biomarker panel which allows the early and accurate diagnosis of sepsis will be developed and optimized. This new panel will also be used to validate the cavity enhanced devices.
Sepsis is a life-threatening illness caused by the body’s overreaction to an infection and can be triggered either directly by infection or may occur after medical treatment or surgery. The immune system reacts against pathogen released molecules (PAMP) [1], indiscriminately whether the pathogen is still alive or has been killed by medical treatment and as a result can cause collateral organ and tissue damage, leading to severe permanent injuries or quite often death. The mortality rate from severe sepsis is 28-50% [2] and it remains the most prevalent cause of death in non-coronary Intensive Care Units (ICUs). It has been estimated that worldwide 20,000 people die daily from sepsis, corresponding to annual death rate of ~530,000 people within the EU [3]. The annual cost of treatment as a result of the hospitalization for sepsis in the US was estimated at $16.7 billion [3]. Diagnosis for treatment currently is through blood culture, which takes 24-72 hours. Every undiagnosed hour increases the mortality risk by 6-10% [4] and so early diagnosis and prompt action is vitally important to reduce mortality, ensure accurate diagnosis, prescribe appropriate treatment, reduce costs and hence minimize the threat to the patient.
There is an urgent need therefore for more rapid diagnosis of a wide range of pathogens, biomarkers and PAMPs as an aid for sepsis diagnosis. In 2010 Pierrakos [5] reviewed the current literature and identified that around 180 molecular markers had been attributed to sepsis. He concluded that using just the prominent markers, C-reactive protein (CRP) and procalcitonin (PCT) has limitations for sepsis diagnosis and that additional markers should be added into a panel to increase specificity as well as sensitivity. Common detection methods include Enzyme Linked Immunosorbant Assays (ELISA) which are typically incorporated into microplates allowing 96 or 384 different samples to be assayed. These tests are currently limited by the choice of markers but also by the sensitivity of the detection instrument. Fluorescence and luminescence based techniques are more sensitive than those using absorbance, but are also more expensive and fewer markers are available. A generic absorbance technique that has the required sensitivity but is of lower cost would be highly desirable. Analysis for additional markers requires more tests to be performed and so the use of more microtiter plates leading to increased material consumption and costs. In addition more sample material has to be taken from the patient.
Microarrays offer an alternative to a proliferation of microtiter plate based tests. A microarray is essentially a surface with a discrete pattern of spots of different immobilized biomolecules contained within a small area. Each spot consists of a different biomolecule and enables separate analysis of a different target or marker. As such a microarray can be described as miniaturized microplate without wells. Thousands of tests per sample have been demonstrated. Typically detection is made through fluorescence, due to the low sensitivity of absorption detection. Given the need for more markers for sepsis diagnosis [6] it would seem worthwhile to investigate the conversion of microtiter plate based assays for sepsis diagnosis into a microarray format and also methods to increase the sensitivity of absorption detection.
Clinical diagnostics and immunoassays:
The size of the global clinical diagnostics market in 2010 was ~$42 billion [7]. Currently, immunoassays are a well established bioanalytical method in clinical laboratories enhancing diagnostic and medical research methods by facilitating the measurements of a large number of biologically important analytes. The availability of reliable commercial kits often makes them the preferred method of choice, even where analytes can be feasibly determined by other types of assay systems (e.g. chromatography). ELISA (Enzyme-Linked Immunosorbent Assays) are the most widely used type of immunoassay. Currently, the main challenges are related to the design of the assay. These include:
- Better specificity of the immunoassay as this will produce fewer interferences from other biomarkers. (Requires better design of immunoassays).
- Greater accuracy of the tests as this will lead to fewer misdiagnoses. (Requires a larger panel of markers and test format which allows high volume of tests quickly).
- Quicker speed of response and lower limits of detection, as these would allow earlier diagnosis which can be crucial in diseases such as sepsis. (Requires better designed immunoassays, high throughput of tests and more sensitive detection).
Therefore an ELISA based test which uses a panel of markers and is performed in a microarray format can form the basis of a test which is quicker, more accurate and more specific.
Improving the sensitivity of colorimetric detection:
The most commonly used detection method for ELISA tests is based on absorption of a coloured product and usually referred to as colorimetric detection. It is the simplest detection method and also the cheapest, hence its popularity, but it is not as sensitive as either fluorescence or chemiluminescent methods. Therefore it is important to investigate methods which can lead to more sensitive absorption measurements as this would still allow the use of colorimetric detection for tests which require higher sensitivity. Recent methods have improved the sensitivity of absorption measurements through the application of cavity enhanced techniques. These increase the absorption by using multiple reflections of light through the sample, building up a much longer path length and hence much increased absorption values. To date these methods have mainly been applied to gas phase analytes but if they could be applied to the colorimetric detection of ELISA tests in microtiter plates and microarrays, they could greatly improve the sensitivity of measurement.
The CE-microArray project concept:
The CE-microArray project will address the two main barriers preventing the use of colorimetric immunoassays for the diagnosis of disease such as sepsis, namely the sensitivity of the test and the accuracy of the test. Firstly it will lead to the development of microtiter plates and microplate readers which are able to make more sensitive colorimetric measurements as a result of a novel application of cavity enhanced absorption spectroscopy. It will also result in a development of a more specific and accurate immunoassay for sepsis diagnosis based on the use of a new panel of markers and optimization of the immunoassay using a novel method for observing the binding kinetics through label free detection. The new methods will be validated by comparison with clinical samples and standards and will also be generally applicable to other clinical diagnostic methods based on colorimetric immunoassays.
CE-microtiter plates and microplate readers:
The CE-microArray project will develop cavity enhanced microtiter plates and microplate readers. These will allow at least a fivefold increase in sensitivity over conventional colorimetric plate readers.
- The simplest device will consist of custom 96 well microtiter plates with high reflectivity dielectric mirrors deposited on the underside and the coverplate, creating an integral optical cavity. This arrangement will allow the microtiter plates to be used in conventional plate readers.
- A second development will integrate dielectric cavity mirrors into the optical path of a conventional colorimetric microplate reader. This will allow the device to be used with standard microtiter plates and result in an improvement in sensitivity of between 30 and 50 fold.
- The final device will combines the integrated cavity mirrors with an array detector to produce a cavity enhanced imaging microplate reader, capable of making simultaneous colorimetric measurements on a glass microarray with a sensitivity enhancement of greater than 5 fold.
Sepsis immunoassay development and optimization:
The CE-microArray project will develop and optimize a novel marker panel for the diagnosis of sepsis.
[1] M. E. Bianchi, Journal of Leukocyte Biology, 81(1), 1 (2007)
[2] R. Daniels, J. Antimicrob Chemother, 66(2), 11 (2011) and references within
[3] Angus DC, Linde-Zwirble WT, Lidicker J, et al: Epidemiology of severe sepsis in the United States: Analysis of incidence, outcome, and associated costs of care. Crit Care Med, 29, 1303 (2001)
[4] J. Soong, and N. Soni, "Sepsis: recognition and treatment." Clinical medicine 12 (3) 276 (2012)
[5] C. Pierrakos, Critical Care, 14(1) 15 (2010)
[6] P. Angenendt, Drug Discovery Today, 10(7), 503-511, (2005)
[7] PricewaterhouseCoopers clinical diagnostics market report (www.pwc.com/diagnostics2011)
Project Results:
CE-microArray was a two-year programme (extended by a total of eight months via a five-month suspension and three-month extension period) which was structured around technological developments related to cavity enhanced microplate readers and the integration of these to create novel prototype cavity enhanced microplate readers which would then be validated using a novel bioassay for sepsis risk detection.
Within the CE-microArray project the work was divided into 7 workpackages (WP). This comprised four RTD workpackages one for validation, one for dissemination and exploitation, and one for project management. The results from the science and technology workpackages (WP 1-5) are described below. Full details of the deliverables are provided in the submitted reports for the individual deliverables.
WP 1 Cavity enhanced microtiter plates
WP1 involved the design and production of novel cavity enhanced microtiter plates and required the identification of polymer microtiter plates suitable for dielectric mirror deposition followed by mirror deposition. There were 2 deliverables associated with this workpackage.
D1.1: Polymer microtiter plate with fractional optical losses of <0.02 per pass
D1.1 aimed to identify high optical quality injection moulded polymer microtiter plates with fractional optical losses of <0.02 per pass.
Polystyrene Falcon® and Nunc™ glass bottom microtiter plates were identified as standard high quality plates with fractional optical losses of < 0.02 (<67%). The glass bottomed microtiter plates, introduced a loss of ~ 8.5% compared to the ~ 52% loss of the lower optical quality polystyrene microtiter plates at 450 nm that is the wavelength of interest for our bioanalytical measurements.
The plates were sent for e-beam deposition and successfully accepted the dielectric coating by using only ambient temperature (without ion assistance – non-IAD). Using a non IAD deposition will lead to reduced process temperature and lower manufacture cost.
D1.2: Microtiter plates with dielectric mirrors deposited on the bottom and on the coverplate
Polystyrene Falcon and Nunc glass bottom microtiter plates were identified as standard high quality plates with fractional optical losses of < 0.02 (<67%) for D1.2. The plates underwent further tests for biochemistry and thermal stress to investigate their suitability for the coating process. Using only the ambient temperature e-beam thermal evaporation technique by YPT the plates successfully accepted the dielectric coating and had a bandwidth around 100 nm centred at either ~450 nm or ~550 nm. Further measurements by TEES showed that the deposited mirror reflectivity was close to 99% and exhibited relatively low optical absorption. The technique appears to be suitable for polymeric materials, it shows reduced process temperature, lower manufacture cost and simpler deposition.
The results on the cavity enhanced plates produced a ~5 fold improvement in sensitivity which is significantly better than a conventional single pass measurement. Further investigation on the alignment and optical quality of plates and covers can in principle lead to a ~10 fold enhancement over commercial colorimetric plate readers.
WP 2 Bioassay implementation and microarray
WP 2 involved the design of bioassays to provide both a more accurate test for sepsis diagnosis and also a means of testing the performance of the cavity enhanced devices produced in WP3 and WP4. There were 4 deliverables associated with this workpackage.
D2.1 Selection of panel of markers
Identifying biomarkers for the diagnosis of sepsis has been subject to discussion and disagreement in recent years. Distinguishing between sepsis and non-infectious inflammation remains a key challenge, and there are hundreds of discussed potential biomarkers for sepsis.
However, as the CE-microArray project intends to benchmark with existing systems, commercialized “standard markers” are aimed for. A standard device for immune diagnostic at University Clinics Ulm, and against which the CE-microArray devices would be compared for accuracy and sensitivity, is the Siemens IMMULITE. D2.1 therefore represented the first step in the development of the devices and their eventual validation.
After consulting and in accordance with Prof. Schneider at University Clinics Ulm, eight markers were chosen as markers for CE-microArray. They cover a large sepsis panel in terms of functionality and are applied at University Clinics Ulm for sepsis diagnostics. These markers also cover several assay challenges as the thresholds of healthy state vary from some pg/ml (IL-1β, IL-6, IL-10, TNF-α) over several tenth pg/ml (IL-8) to µg/ml (LBP). Enzymatically active markers are also included from mU/ml (EPO) to kU/ml (sCD25).
D2.2 Preparation of reference materials
The aim of the deliverable was the preparation of calibrator solutions mimicking an HRP-ELISA reaction. There were three levels of calibrator solutions planned:
a. Static calibrators: mimic an endpoint measurement and are a dye solution
b. Dynamic calibrators: mimic the staining reaction and are a two compound system containing an HRP-active part and a dye part
c. Dynamic handling calibrators: mimic the last step of an ELISA and provide binding moieties to the surface, followed by a one-step binding of a HRP active compound. In accordance with the aims, several commercial easy available dyes were tested for application as static calibrators. The dynamic calibrators were based on streptavidin-HRP and TMB. The dynamic handling calibrators had in addition biotinylated BSA.
The calibrator solutions will enable the partners to run their own measurements independent of the status of the sepsis marker panel. This will decouple the workflow and allow each partner to succeed independent of the results from any other. As the calibrators are benchmarked to IL8 each partner can measure the sensitivity of the according system by also applying it to any other microplate reader capable to read wavelength at 480 or 600 nm.
D2.3 Validation of panel of markers in microplate format
The aim of the deliverable was validation of a panel of markers in microplate format and provision of calibration solutions to project partners.
In accordance with D2.2 a total of 8 biomarkers were selected and according antibodies will be tested. In terms of cost and availability it is in general more cost-efficient to buy several antibodies and test them for compatibility with ELISA, but the success rate can be quite small and the smaller price comes in terms of cost in time as it can take several month to improve a binding pair to diagnostic sensitivity. In a first attempt this was applied to the marker IL8, but it became clear that for the other markers it is more time-saving to simply order the according markers, even if the number of measurements is then drastically limited within the process. In addition it is not possible with such kits to measure the binding kinetics via iRIF as the amount of material on these kits is so small, that an iRIF-measurement would cost around 1,500 EUR per run. Therefore we made for the validation of the IL8 and some other panel markers iRIF-measurements to derive some protocol improvements. As we later applied the complete sets, we could not use the iRIF-setup anymore and simply used the vendor protocols for sake of high sensitivity. From the previous selected 8 biomarkers one failed completely, but the other 7 could be completely established in the lab for measurements in a microplate.
Therefore the deliverable D2.3 was met in all terms, that a full validation in a microplate format was shown and is compatible with the CE-microArray reader. For the purpose of the compatibility Dr. Zuzana Bajuszova from Teesside University visited Freiburg to make jointly the HRP-reaction for detection as well as microcontact printing of structures. This knowhow transfer ensures the compatibility from MTP to the CE-microarray reader.
With this step the validation of the CE-microArray devices against pre-characterized clinical samples in D5.3 can be realized, as now the antibodies and protocols are available for the testing.
D2.4: Testing of chemical protocol for antibody immobilization
D2.4 aimed to establish assay protocols for the biomarkers identified in D2.2. In D2.2 a total of 8 biomarkers were selected and validated in D2.3 accordingly.
It was possible to show how to optimize an assay system applied to the marker IL8, but it became clear that for the other markers it is more time-saving to simply order the according markers, even if the number of measurements is then drastically limited within the process. In addition it is not possible with such kits to measure the binding kinetics via iRIF as the amount of material on these kits is so small, that an iRIF-measurement would cost around 1,500 EUR per run. Therefore we made for the validation of the IL8 and some other panel markers iRIF-measurements to derive some protocol improvements. As we later applied the complete sets, we could no longer use the iRIF set-up and simply used the vendor protocols for the sake of high sensitivity. From the previous selected 8 biomarkers one failed completely, but the other seven could be completely established in the lab for measurements in a microplate.
WP 3 Prototype cavity enhanced microplate reader platform
The overall goal of WP3 was to develop two novel prototype cavity enhanced microplate readers which would be used to make cavity enhanced measurements on standard 96 well microtiter plates and also microarrays. It consisted of three deliverables.
D3.1: A prototype cavity enhanced microplate reader
The deliverable seeks to develop a prototype cavity enhanced microplate reader capable of measurements on single wells of a 96 well micotiter plate.
A fibre-coupled vertical optical cavity was developed and further integrated with a motorized XY stage to form the backbone of the commercial grade instrument in D4.1. The stage functioned as a receptacle for the microtiter plate and allowed cavity enhanced point measurements on individual wells. Fibre-coupling provided the advantage of a relatively easy to align optical setup with well collimated light beam and simple setup geometry.
Task 3.1 also involved the investigation of the effect of the meniscus and the optical quality of the microtiter plates on the optical losses. To minimise losses due to liquid/air interface two approaches were implemented, involving either dosing exact amount of sample fluid for full well capacity or using high quality coverplates covering the surface of sample wells. While accurate filling is likely to be the simplest approach, coverplates have the advantage of ensuring a stable flat fluid interface.
Polystyrene Falcon® and Nunc™ glass bottom microtiter plates were identified as standard high quality plates with fractional optical losses of < 0.02 in D1.1. Their optical quality as part of Task 3.1 was investigated by recording the introduced cavity losses and obtaining the values of cavity enhancement. The insertion of the glass bottomed plate into the cavity with a blank solvent solution provided a loss of ~ 48%. The relative loss by the polystyrene plate due to lower optical quality was ~ 60%. The presence of a coverplate introduced an additional ~ 6% loss. This resulted in a cavity enhancement value of 37 for glass and 23 for polymer bottom plates. The enhancement increased to 58 fold and 41 fold respectively with the method of accurate filling.
The results produced substantial improvement over conventional measurements and met D3.1 aiming to produce a prototype CE-microplate reader with a cavity enhancement factor of > 30. A schematic of D3.1 is shown as figure 1.
D3.2: A prototype cavity enhanced imaging microplate reader capable of multiplexed detection
Originally, the aim of D3.2 was to develop a cavity enhanced imaging plate reader using a point by point scanning in raster scan mode. This would have involved moving the motorised XY stage over small area and simultaneously recording the spectra. However as the developmental work progressed we realised that this approach of acquiring a cavity enhanced image would be relatively time consuming and not the best use of our resources. Therefore the consortium decided to propose a change in the deliverable and produce and interim multiplex prototype for D3.2 which would be further developed in D3.3.
The prototype constructed was based on a 2×2 array of individually assembled dielectric mirrors, collimators and multi-furcated optic fibres. The output was monitored with a multichannel spectrograph allowing two well to be monitored simultaneously.
The experimental work involved obtaining the absorption spectra of two independent set of measurements at the same time, using a single light source, an array of mirrors, a microtiter plate and a single detector. Results showed a CEF value of 30 for channel 1 and a value of 29 for channel 2. The agreement within the values indicates a well aligned array of optical cavities and high accuracy in manufacturing.
Overall, we can conclude that the construction of the intermediate prototype for multiplexing was successful and will be the foundation of the multiplex cavity enhanced plate reader using an imaging spectrometer in D3.3
D3.3: A prototype cavity enhanced imaging microplate reader using a 2D array detector
D3.3 sought to develop a prototype cavity enhanced imaging microplate reader by incorporating a camera into the cavity enhanced optical setup, hence providing a recorded cavity enhanced image with at least 30 fold improvement compared to conventional imaging plate readers.
A free-space coupled CE-imaging plate reader was developed, where a Point Grey monochrome camera operated in 16-bit mode was used to record the images. To identify the most compatible arrangement with imaging, we decided to investigate three cavity configuration. For demonstration purposes and ease of mounting the array was tested on optically clear microscope slides. Key scientific and technical challenges existed in constructing this prototype as it represented a truly novel area with little previous work. During the course of development we established the experimental methodology to obtaining such images and processing them to provide a valid end result. The CEF value contained contributions from surface related scattering losses and was a measure of the total cavity loss. The slides contributed in average 14% loss to the system and were held in a custom made slide holder.
The first optical cavity was formed by two plane mirrors with average reflectivity of 94.5%.This configuration is highly unstable and required a well aligned optical cavity with a well collimated light beam incident on the output mirror. To increase the stability of the system the mirrors were separated by the minimum distance available. The recorded cavity images yielded to a significant colour enhancement and provided 11 fold improvement when compared to conventional imaging. This geometry had no focusing point hence provided a relatively large beam area of ~5 mm in diameter enable to contain a 2×2 array.
The second configuration was formed by one plane and one concave mirror of average reflectivity of 99%. This arrangement is relatively stable but due to the presence of a focusing mirror provided a relatively small centre area of 1 mm in diameter available for imaging. This resulted in reducing the size of the manually spotted array. The geometry also required a well-positioned array through the centre line of the beam to avoid side reflections. The results showed a 27 fold enhancement that was further improved to 43.
The final configuration was the most stable from all three tested, containing two confocal mirrors of 99% reflectivity. Similar to the previous arrangement the presence of the focusing mirror yielded a centre hot spot, requiring a well-positioned array at the centre line of the beam. This introduced additional challenges to the alignment procedure. The resulting cavity enhanced images yielded to a 30 fold improvement when compared to conventional single-pass imaging.
Overall we can conclude that the construction of a CE-imaging prototype was successful and D3.3's proposal of an enhancement of >30 was met. The intention was to proceed with a plane-parallel configuration for D4.2 as it provides a large area for the array, a simpler image processing methodology and has the potential to provide a cavity image with >30 fold enhancement by the use of custom made 99% mirror set.
WP 4 Integration of reader with bioassay
The main aim of WP 4 was to take the prototype lab based cavity enhanced microplate readers developed in WP 3 and engineer them into commercial grade cavity enhanced microplate readers. Some of the bioassays developed in WP 2 were also tested and optimised for use in the microplate readers in this workpackage. Unexpected technical challenges arising for the construction of the prototypes for D4.1 and D4.2 led to a project suspension of five months and a project extension of three months to allow the deliverables to be successfully completed. WP 4 consisted of three deliverables.
D4.1: A commercial grade cavity enhanced microplate reader
The aim of D4.1 was to convert the prototype cavity enhanced microplate reader capable of measurements on single wells of a 96 well microtiter plate, developed in D3.1 into a commercial grade instrument. This work was carried out by consortium partners EII in collaboration with TEES. A stand alone cavity enhanced microplate reader was developed along with the custom software required to operate the instrument and analyse the recorded spectra. A render of the instrument is shown in figure 2. Full details of the mechanical operation of the instrument and the functionality of the software can be found in the submitted report for D4.1.
D4.2: A commercial grade cavity enhanced imaging microplate reader
The aim of D4.2 was to convert the prototype cavity enhanced imaging microplate reader capable of imaging measurements, developed in D3.3 into a commercial grade instrument. This work was carried out by consortium partners EII in collaboration with TEES. A stand alone cavity enhanced imaging microplate reader was developed along with the custom software required to operate the instrument and analyse the recorded spectra. A render of the instrument is shown in figure 3. Full details of the mechanical operation of the instrument and the functionality of the software can be found in the submitted report for D4.2.
D4.3: Tested and improved assay protocols and calibration samples to benchmark readers
The aim of D4.3 was to develop calibration samples and improved assay protocols (ALU-FR) which would be used later on in D5.2 to test the performance of the cavity enhanced plate readers developed in D4.1 and D4.2.
As such ALU-FR provided the knowledge and handling of the standard microarray readers and developed a bBSA as well as a HRP binding assay enabling comparison between the microplate readers used in ALU-FR – a victor reader V3, Berthold technologies, Germany – and the CE microplate readers later on in D5.2.
WP 5 Validation
WP5 aimed to validate the cavity enhanced devices produced by the CE-microArray project. Some of the validation work undertaken within this workpackage was subcontracted as per the DoW to University Clinics Ulm (UCU) (Germany) headed by Prof Marion Schneider as they have great expertise in the diagnosis and treatment of sepsis and related infections. There were four deliverables associated with this workpackage.
D5.1: Ethical clearance
Ethical clearance was obtained the Ethics Commission of the University of Ulm to use anonymised patient data from ongoing clinical studies with intensive care patients as a platform to perform the project’s validation work. An ongoing study was extended, and confirmation was provided by the University of Ulm that the tests wwill use samples from those who have given consent for their samples to be used for the purposes of commercial test development.
D5.2: Evaluation report comparing CE-microArray devices with standard microplate assays
D5.2 sought to validate the CE-microArray devices against calibration samples. Successful application was made of the D4.1 CE-microArray 96 well Plate Reader to standard colorimetric assays and the D4.2 CE-microArray Imaging Plate Reader to bio printed arrays.
For the first test series, a standard STREP-HRP colorimetric assay and a standard Osteocalcin ELISA assay was performed, and the signal at 450 nm was read on the CE-microArray 96 well plate reader. The results were benchmarked against a standard microplate reader and showed 77-91 fold improvement in sensitivity.
In the next test, the CE-microArray Imaging Plate Reader was tested against a microcontact printed bio array (assay developed by ALU-FR) on a glass surface and the results were benchmarked against conventional images taken by the reader. The results showed 6-8 fold enhancement compared to the images taken without the presence of the cavity mirrors.
We can conclude that the validation of the CE-microArray devices against standard colorimetric assay were successful and the proposed enhancement of >30 for prototype D4.1 and enhancement >5 for prototype D4.2 was met.
The final step was to involve the validation of the CE-microArray devices against pre-characterized clinical samples in D5.3.
D5.3: Evaluation report comparing with clinical samples
The aim of D5.3 was to perform comparative measurements on clinical samples using the cavity enhanced microplate readers and a standard microplate reader. This required making the measurements at University Clinics Ulm (UCU) (Germany) under the supervision of Prof Marion Schneider as UCU has the facilities to safely make measurements on clinical samples. Dr Günter Roth from ALU-FR travelled to Ulm with test calibration assays. The cavity enhanced microplate reader was sent by TEES and Dr Zuzana Bajuszova (TEES) travelled to Ulm to make the measurements with the cavity enhanced microplate reader. Unfortunately when the cavity enhanced microplate reader was unpacked in Ulm it was found to have been damaged in transit and was inoperable. This led to the planned comparative measurements having to be cancelled. Repair of the instrument was not possible onsite, would require the visit of engineers from EII and would require several weeks to achieve. As these measurements were planned for the end of the project there was insufficient time remaining to reschedule the measurements. However, from the measurements performed for D5.2 it is possible to estimate that the cavity enhanced microplate readers would be approximately 30 times more sensitive for the D4.1 prototype and 6-8 more sensitive for the D4.2 prototype.
Technicians at Ulm subsequently made some repairs to D4.1 and it will shortly be returned to the UK for realignment. The consortium partners were updated of this situation, and confirmation was received from SME partners ANX, ASYS, BMT, NEB and YPT of their ongoing interest to continue to work together to develop the project results despite this setback (the SMEs are due to form the Joint Venture Board to after the project ends). This is felt to reflect the longer-term value of the project work and its results.
D5.4: Establishing an ex-vivo whole blood stimulation system
To understand the nature of immune dysfunction in patients with sepsis manifesting after severe trauma, a robust whole blood ex vivo test system (using TruCultureTM) was developed as D5.4.
One millilitre of blood was drawn into separate syringes containing ligands to stimulate the following TLR2-9. Amongst many others, the biomarkers TNF-α, Interleukin-1β and TNF-α followed by IL-1RA and soluble TNF-RII was quantified by multiplexed sandwich immunoassays. The IL-1ratio and TNFratio following stimulation with LPS, were defined as the ratios of IL-1β [pg/ml]/IL-1RA [pg/ml] and TNF-α [pg/ml] /sTNF-RII [pg/ml], respectively.
When compared with healthy donors, most TLR induced cytokines were lower in trauma and even lower in sepsis patients’ cultures. An exception was the TLR2 stimulation, which induced more inflammatory and anti-inflammatory cytokines as well as soluble receptors in trauma and sepsis than in healthy donors. Among the other TLR responses, TLR3 was most dramatically downregulated in patients with trauma and even more in sepsis patients. Calculating the IL-1ratio and the TNFratio, we found a patient-type specific ratio of ligand to antagonists. Healthy donors had a median IL-1ratio of 1.48 and a median TNFratio of 2.73 trauma patients had 10times -, and sepsis patients had 100times lower IL-1 and TNFratios.
The developed TruCultureTM ex-vivo whole blood TLR stimulation test is valid to correlate a defined response pattern to the clinically established stages of trauma and sepsis. Results may substantially contribute to signalling pathways leading to immune dysfunction. To link to WP2, the biomarkers identified within WP2 were measured in D5.4.
Potential Impact:
Potential Impact:
The potential impact of the work is based on the further development and commercialisation of the project results:
Result 1, Microtiter plates with dielectric mirrors deposited on the bottom and on the coverplate, which have the potential to be commercialised as a consumable which gives significantly improved sensitivity compared to a normal plate.
Result 2, Tested chemical protocol antibody immobilization and assay protocol, which will require further research, development and validation, leading to further testing and clinical trials. This is expected to take at least a further two years.
Results 3 and 4, prototype and commercial-grade cavity enhanced imaging microplate readers, and the Cavity Enhanced Microplate Reader – these will be positioned as highly sensitive microplate readers.
Result 5, Establishing an ex-vivo whole blood stimulation system, which will link to result 2 and will contribute to the aim of developing diagnostic tests for sepsis.
The main focus is on the commercial impact which will result from further developing the results and taking them to market, and also the wider societal implications which would result.
In terms of economic impact, the successful commercialisation of the results would result in increased sales, turnover and potential job creation within the consortium companies.
In terms of broader societal impact, the ultimate aim of the project is to develop a more sensitive, accurate, faster and more useful diagnostic platform for sepsis, and to help to provide patients with early, accurate diagnosis. Sepsis is the most prevalent cause of death in non-coronary Intensive Care Units (ICUs), and the mortality rate in patients admitted to hospital with severe sepsis is 28-50%. The potential impact of the project in helping to address this situation is therefore substantial, in terms of improving mortality rates, supporting healthcare efficiency and reducing costs. It has been estimated that in the US ~$17 billion is spent annually treating sepsis in patients and that a similar amount is spent across the EU (Angus et al, 2001).
Patent searches have been undertaken as part of the preparation of the interim and final Plans for the Use and Dissemination of Knowledge, and nothing has been found which would restrict the consortium’s freedom to operate and to commercialise / exploit the project results.
As explained in several Deliverable reports including the Final Plan for the Use and Dissemination of Knowledge, it will take an estimated two more years for additional testing and clinical trials of result two (the tested chemical protocol antibody immobilization and assay protocol) as part of the integration of the project results into a diagnostic platform. In the shorter-term, impact will be realised from the further development and progress towards commercialisation of results one, three and four.
Result one has the potential to be commercialised as a consumable which offers increased sensitivity compared to standard plates.
For the Cavity Enhanced Microplate Reader and the Cavity Enhanced Imaging Microplate Reader, the partners will target more broadly the microplate reader market, including as medical diagnostic equipment. The key decision factor when purchasing a new plate reader is sensitivity, and there is a large market for low cost, highly sensitive absorbance based microplate readers. D6.2 the Market Report, showed that the annual global market for microplate readers was estimated to $300 million according to the survey in 2009 (Banks, 2009).
D6.2 also outlines that the lifetime of the proposed reader will be at least five years, based on the relatively long expected lifetime of its individual components. By providing an increased sensitivity over standard absorption microplate readers at the same price point, the consortium aims to achieve a 1% market share in the single mode absorbance microplate reader and absorbance based imaging microplate reader markets.
D4.1 concludes that the validation of the CE-microArray devices against standard colorimetric assay was successful, and that the proposed enhancement of greater than 30-fold for prototype D4.1 and greater than 5-fold for prototype D4.2 was met. This provides a strong basis for the realisation of the project’s impact potential.
Main Dissemination Activities:
The main dissemination activities which have been carried out so far are as follows.
Activity aimed at an industrial or end-user audience:
- ASYS showcased the project results at the PHOTONEX 2016 exhibition in Coventry, UK, on 12th and 13th October 2016. This is a trade show for photonics and light-based technologies, with an audience of industrial representatives from the UK and overseas.
- The promotional pamphlet developed during the project as D6.6 was printed and taken to the PHOTONEX exhibition to be handed to delegates.
- TEES was one of 23 organisations which took part in a visit to India in May / June 2016. This was led by the Chairman of NHS England and aimed to showcase research and innovation with healthcare applications, and to develop collaborative links. CE-microArray was part of the research showcased by TEES during this visit.
- A promotional video (duration 1 minute, 53 seconds) was produced as part of the visit to India to provide information on the aims and work of CE-microArray. This shows prototype D4.1 being used in the TEES laboratories.
- A press release, available at
http://www.tees.ac.uk/sections/research/news_story.cfm?story_id=4608&this_issue_title=March%202014&this_issue=250, was issued by Teesside University at the start of the project and subsequently formed the basis of articles about CE-microArray in, amongst others, LabMate Online (based in the UK), Nursing Standard (UK), Pharmiweb.com (UK), Pharma Focus Asia (India), PR Newswire UK, Wallstreet: Online (Germany), Informazione.it (Italy), and local newspapers and radio stations in Middlesbrough and the north-east of England.
- An entry for the project was created on the research section of the Teesside University, ALU-FR and EII websites respectively, alongside overviews of other projects and activity.
The website was updated during the project and a total of 19,625 hits from 4,295 visits were recorded over the project lifetime (to 23rd November 2016) by the project website platform, iPage. The Deliverables which were identified at the start of the project as ‘Public’ (i.e. not commerciality sensitive or confidential) have been made available on the website. These are the promotional leaflet (D6.6) and the report on regulatory and standardisation requirements in EU and US markets (D6.5).
The main dissemination activities to a scientific audience are:
Two journal papers were published:
- Bajuszova, Z., Ali, Z., Scott, S., Seetohul, L. N., Islam, M. (2016) 'Cavity Enhanced Immunoassay Measurements in Microtiter Plates using BBCEAS' Analytical Chemistry; 88 (10): 5264-5270, DOI 10.1021/acs.analchem.6b00375.
- Burger, J., Rath, C., Wöhrle, J., Meyer, P.A. Anmar, N.B. Kilb, N., Brandstetter, T., Pröll, F., Proll, G. Urban, G., Roth, G. (2016), ‘Low-Volume Label-Free Detection of Molecule-Protein Interactions on Microarrays by Imaging Reflectometric Interferometry’ Journal of Laboratory Automation, DOI 10.1177/2211068216657512.
One conference presentation was given by the project coordinator, Prof. Meez Islam, who presented the project during a talk on ‘Novel Applications of Liquid-phase CE Absorption Spectroscopy’ at the Cavity Enhanced Spectroscopy (CES) conference, which was held in Boulder, Colorado, USA, June 16th – 19th 2015.
Further dissemination activity is planned as follows for the initial period after the project:
Journal articles:
- A journal article on the development of result 1, the microtiter plates with dielectric mirrors deposited on the bottom and on the coverplate, and the increase in sensitivity achieved with the plates. This will be co-authored by TEES and YPT.
The target journal for this paper is Analyst, which is published by the Royal Society of Chemistry in the UK and supports Open Access publishing via either Gold or Green routes (after a 12-month embargo period in the case of Green Open Access).
- Two journal articles on cavity enhanced imaging, the preparation of which will be led by TEES. The target journals are Optics Express, which is published by the Optical Society of America and also support Open Access via Gold or Green routes (with a 12-month embargo period for Green Open Access); and PLOS ONE, which is published by the non-profit published PLOS, and which supports Open Access publishing with no embargo on Green Open Access.
Exhibitions / trade shows:
- ASYS will follow-up from contacts made at the PHOTONEX exhibition in October 2016 – several companies expressed an interest in the project devices, and it would be beneficial to develop these contacts further (although as stated in section 3.1 further development work will be needed before commercialisation can proceed).
- The potential of attending European trade shows will be explored, notably MEDICA, the World Forum for Medicine, which takes place in Düsseldorf in Germany. Due to the respective stages of development of the project results, and the need for further development work of results two and five, the initial focus would be on the Cavity Enhanced Microplate, the Cavity Enhanced Microplate Reader and the Cavity Enhanced Imaging Microplate Reader. Partners will also carry out an overview of other relevant trade shows to identify possible targets as the development of the project results progresses.
The project website will also be maintained following the end of the project.
A significant focus of dissemination activity in the period immediately following the end of the project will be to build links with other companies whilst continuing to develop the project results. Partners’ current contacts and networks will be utilised to raise awareness of the results, and opportunities will be taken to meet company representatives face-to-face. It is anticipated that this may lead to the development of collaborative activity, including the development of additional applications for external funding to further develop the project results.
Exploitation of Results:
The aim is to develop the following three products:
1. Cavity Enhanced Microplate (result 1)
A modified microplate with integral dielectric mirrors to enhance the operation of microplate readers.
2. Cavity Enhanced Microplate Reader
A diagnostic tool made to perform cavity enhanced absorption measurements on a standard 96 microwell array.
3. Cavity Enhanced Imaging Microplate Reader (result 3, 4)
A more complex diagnostic tool incorporating a 2D detector capable of making multiplex imaging measurements on 96 well microtitre plates.
The aim is also to further develop result 2, a tested chemical protocol antibody immobilization and assay protocol, result 5, and an ex-vivo whole blood stimulation system.
As stated above, the overall objective is to combine these products to address the ultimate goal of developing a more sensitive, accurate, faster and more useful platform for the diagnosis of sepsis. As results 2 and 5 are expected to take a longer time to develop, emphasis will be placed in the intervening period on developing the Cavity Enhanced Microplate, the Cavity Enhanced Microplate Reader and the Cavity Enhanced Imaging Microplate Reader for other markets as they are closer to commercialisation, whilst maintaining a focus on the overall goal.
The Final Plan for the Use and Dissemination of Knowledge sets out pathways towards the commercialisation and exploitation of the results. These are as follows:
Result 1:
This result is to be positioned as a consumable. This project result will require further development work, as the process which has been developed within the project to deposit the mirrors is relatively expensive. A lower-cost process would be required to position this as a competitively-priced consumable. The current process also takes a relatively long time, and is limited to the production of batches of plates, rather than a more advantageous continuous process.
In terms of advantages, this project result has the potential to fill a significant gap in the market, as in the partners’ considerable experience of this market, no similar plate is currently available. Initial measurements collected as part of the project indicate that the plates give significantly improved sensitivity compared to a normal plate, which has the potential to be appealing within the marketplace.
This project result would be marketed as a disposable consumable, so has the potential to result in a strong revenue stream.
Further funding is needed to explore and optimise the process of producing the plates. This would take more time and concentrated effort than has been available in CE-microArray.
In the longer-term, a partnership may be required with a large manufacturer to make the plates on a sufficiently large scale.
Prospective lead partners: YPT, UP, working with ASYS
Result 2:
As stated in D6.4 further research, development and validation will be required after the project to further develop result 2, leading to further testing and clinical trials. This is expected to take at least a further two years. This is particularly the case as project resulted in a smaller number of antibody pairs than was planned at the start of CE-microArray.
Also as stated in D6.4 result two has led to the additional benefit of supporting BMT in extending their iRIfS technology and its uses and applications.
Further funding and investment will be necessary, targeting national or European funding at first to make more progress before using the results to target commercialisation via large clinical diagnostics companies.
The partners have some contacts with these companies, which would add support to funding applications and the later activity.
Prospective lead partners: BMT, NEB, with the support of ASYS
Result 3 and 4:
The aim is to position results 3 and 4, and the Cavity Enhanced Microplate Reader, to meet a market need for highly sensitive microplate readers.
Further development work will be needed after the end of the project – as stated in D7.5 some of the devices’ current components are relatively expensive, and an initial focus will be to reduce the cost by simplifying their construction whilst safeguarding / improving usability.
The aim will be to build links with companies in parallel to this activity in order to demonstrate and raise interest in the results, but it is anticipated that the cost will need to be reduced before the interest of these companies can be secured.
Following this stage, the main options will be:
- for a larger company to purchase the IP and develop the products as part of their own technology / product range. This would rely on finding a company with very strong interest in the results, and it would also end the partners’ ability to further develop them;
- the partners further developing the project results themselves, and to collaborate with a smaller company to sell or distribute them as final products. This would allow the partners to retain greater control and to build their own reputation as the products would carry their branding and become part of their range. This would be a preferable option from the perspective of ASYS as project Exploitation Manager.
More funding will be required to reduce the costs of the prototypes.
Potential targets include the Emerging and Enabling Technologies programme or the Small Business Research Initiative programme, both of which are offered by Innovate UK. The H2020 SME Instrument is another possible target.
In addition, TEES is a partner in the H2020 GateOne project, GA number 644856, which is developing demonstrator technology for SMEs. This may provide opportunities for cross-fertilisation and for the further shared or joint development of the CE-microArray and GateOne products.
Prospective lead partners: ASYS, ANX
Result 5:
This result contributes to the aim of developing diagnostic tests for sepsis, and will be of interest to clinicians. It is based on the use of the existing TruCulture system, so will contribute to the work required to further develop result 2.
The result will be taken forward as part of the development of result 2: further funding will be required for additional clinical testing and validation.
Prospective lead partners: BMT, NEB
In summary, the exploitation of the results has not yet begun in earnest as the focus has been on delivering the technical project work packages and the demonstration work package. In particular, it was not possible to complete Deliverable 6.8 the invitational meeting targeted at producers of diagnostics, as there was simply insufficient time within the project lifespan between the completion of the project results and the end of the project period. It was therefore not possible to undertake advanced dissemination of this type, which would require a longer lead-in time and will more feasibly be carried out after the completion of the project and a period of further development and refinement work. It is felt important that direct dissemination of this kind to a group of companies takes place only when the project devices (D4.1 D4.2) are ready to be presented to and scrutinised by such an audience. It will be vital to take this opportunity to build a strong reputation and ensure as far as possible that a strong impression is made and that good relationships begin to be forged.
In order to gain initial feedback from relevant companies, ASYS showcased project devices D4.1 and D4.2 at the PHOTONEX trade exhibition in Coventry, UK, on 12th and 13th October 2016. This is a trade show for photonics and light-based technologies, with an audience of industrial representatives from the UK and overseas. Although the exhibition took place after the end of the project, the majority of the preparation took place during the project lifetime. Attendance at this exhibition allowed ASYS to make connections with companies who are interested in the project devices, and served as preparation for the more advanced dissemination activity which will take place subsequently.
The results of the technical work packages provide a strong foundation on which to build dissemination and commercialisation activity.
Dissemination activity in work package 6 has therefore mainly focused on preparing for dissemination following the end of the project. This has been done via D6.2 the report on market analysis and potential for microplates and microplate readers; D6.5 the report on regulatory and standardization requirements in EU and US markets; D6.6 the pamphlet for distribution to end-users; and D6.7 the final plan for the use and dissemination of knowledge. These Deliverables will guide dissemination and commercialisation activities following the end of the project.
List of Websites:
Website address: www.cemicroarray.com. Contact details: Professor Meez Islam, m.islam@tees.ac.uk.
The CE-microArray project was a two-year programme (extended by a total of eight months via a five-month suspension and three-month extension period) which had two main aims. Firstly, to increase the sensitivity of detection of colorimetric ELISAs and microarrays by developing novel microtiter plates and plate readers based on cavity enhanced absorption spectroscopy. The secondary aim was to develop a novel bioassay which could be used to improve the sensitivity and selectivity of sepsis risk diagnosis. Sepsis is a life-threatening illness caused by the body’s overreaction to an infection and can be triggered either directly by infection or may occur after medical treatment or surgery. The mortality rate in patients admitted to hospital with severe sepsis is 28-50% and it remains the most prevalent cause of death in non-coronary Intensive Care Units (ICUs ).
The CE-microArray project led to the development of three types of cavity enhanced devices. Firstly, a cavity enhanced microtiter plate which had high reflectivity dielectric mirrors deposited on the underside of the plate and on the coverplate. This led to a self contained cavity enhanced device which was able to provide a five fold increase in the path length and sensitivity of measurement compared to a conventional microtiter plate. The second device was a cavity enhanced microplate reader which was able to make single point measurements in the wells of a standard 96 well microtiter plate with greater than 30 fold increase in sensitivity when compared to measurements with a conventional microtiter plate reader. The final device was a cavity enhanced imaging microtiter plate reader was capable of making imaging measurements on colorimetric microarrays with a 6-8 fold increase in sensitivity.
In parallel to the development of the cavity enhanced devices, consortium partners developed a novel bioassay for sepsis risk diagnosis based on choosing a selection of biomarkers identified by experts in the field and then developing suitable assays for the biomarkers. Calibration samples were also provided to the test the relative sensitivity of the cavity enhanced devices compared to conventional plate readers.
Project Context and Objectives:
The overall objectives of the CE-microArray project were the production of cavity enhanced microtiter plates (MTP) and cavity enhanced microplate readers which will result in up to a 100 fold increase in the sensitivity of colorimetric bioassays, leading to higher specificity for target analytes as well as cost reductions from the use of smaller quantities of biochemicals. In addition a novel biomarker panel which allows the early and accurate diagnosis of sepsis will be developed and optimized. This new panel will also be used to validate the cavity enhanced devices.
Sepsis is a life-threatening illness caused by the body’s overreaction to an infection and can be triggered either directly by infection or may occur after medical treatment or surgery. The immune system reacts against pathogen released molecules (PAMP) [1], indiscriminately whether the pathogen is still alive or has been killed by medical treatment and as a result can cause collateral organ and tissue damage, leading to severe permanent injuries or quite often death. The mortality rate from severe sepsis is 28-50% [2] and it remains the most prevalent cause of death in non-coronary Intensive Care Units (ICUs). It has been estimated that worldwide 20,000 people die daily from sepsis, corresponding to annual death rate of ~530,000 people within the EU [3]. The annual cost of treatment as a result of the hospitalization for sepsis in the US was estimated at $16.7 billion [3]. Diagnosis for treatment currently is through blood culture, which takes 24-72 hours. Every undiagnosed hour increases the mortality risk by 6-10% [4] and so early diagnosis and prompt action is vitally important to reduce mortality, ensure accurate diagnosis, prescribe appropriate treatment, reduce costs and hence minimize the threat to the patient.
There is an urgent need therefore for more rapid diagnosis of a wide range of pathogens, biomarkers and PAMPs as an aid for sepsis diagnosis. In 2010 Pierrakos [5] reviewed the current literature and identified that around 180 molecular markers had been attributed to sepsis. He concluded that using just the prominent markers, C-reactive protein (CRP) and procalcitonin (PCT) has limitations for sepsis diagnosis and that additional markers should be added into a panel to increase specificity as well as sensitivity. Common detection methods include Enzyme Linked Immunosorbant Assays (ELISA) which are typically incorporated into microplates allowing 96 or 384 different samples to be assayed. These tests are currently limited by the choice of markers but also by the sensitivity of the detection instrument. Fluorescence and luminescence based techniques are more sensitive than those using absorbance, but are also more expensive and fewer markers are available. A generic absorbance technique that has the required sensitivity but is of lower cost would be highly desirable. Analysis for additional markers requires more tests to be performed and so the use of more microtiter plates leading to increased material consumption and costs. In addition more sample material has to be taken from the patient.
Microarrays offer an alternative to a proliferation of microtiter plate based tests. A microarray is essentially a surface with a discrete pattern of spots of different immobilized biomolecules contained within a small area. Each spot consists of a different biomolecule and enables separate analysis of a different target or marker. As such a microarray can be described as miniaturized microplate without wells. Thousands of tests per sample have been demonstrated. Typically detection is made through fluorescence, due to the low sensitivity of absorption detection. Given the need for more markers for sepsis diagnosis [6] it would seem worthwhile to investigate the conversion of microtiter plate based assays for sepsis diagnosis into a microarray format and also methods to increase the sensitivity of absorption detection.
Clinical diagnostics and immunoassays:
The size of the global clinical diagnostics market in 2010 was ~$42 billion [7]. Currently, immunoassays are a well established bioanalytical method in clinical laboratories enhancing diagnostic and medical research methods by facilitating the measurements of a large number of biologically important analytes. The availability of reliable commercial kits often makes them the preferred method of choice, even where analytes can be feasibly determined by other types of assay systems (e.g. chromatography). ELISA (Enzyme-Linked Immunosorbent Assays) are the most widely used type of immunoassay. Currently, the main challenges are related to the design of the assay. These include:
- Better specificity of the immunoassay as this will produce fewer interferences from other biomarkers. (Requires better design of immunoassays).
- Greater accuracy of the tests as this will lead to fewer misdiagnoses. (Requires a larger panel of markers and test format which allows high volume of tests quickly).
- Quicker speed of response and lower limits of detection, as these would allow earlier diagnosis which can be crucial in diseases such as sepsis. (Requires better designed immunoassays, high throughput of tests and more sensitive detection).
Therefore an ELISA based test which uses a panel of markers and is performed in a microarray format can form the basis of a test which is quicker, more accurate and more specific.
Improving the sensitivity of colorimetric detection:
The most commonly used detection method for ELISA tests is based on absorption of a coloured product and usually referred to as colorimetric detection. It is the simplest detection method and also the cheapest, hence its popularity, but it is not as sensitive as either fluorescence or chemiluminescent methods. Therefore it is important to investigate methods which can lead to more sensitive absorption measurements as this would still allow the use of colorimetric detection for tests which require higher sensitivity. Recent methods have improved the sensitivity of absorption measurements through the application of cavity enhanced techniques. These increase the absorption by using multiple reflections of light through the sample, building up a much longer path length and hence much increased absorption values. To date these methods have mainly been applied to gas phase analytes but if they could be applied to the colorimetric detection of ELISA tests in microtiter plates and microarrays, they could greatly improve the sensitivity of measurement.
The CE-microArray project concept:
The CE-microArray project will address the two main barriers preventing the use of colorimetric immunoassays for the diagnosis of disease such as sepsis, namely the sensitivity of the test and the accuracy of the test. Firstly it will lead to the development of microtiter plates and microplate readers which are able to make more sensitive colorimetric measurements as a result of a novel application of cavity enhanced absorption spectroscopy. It will also result in a development of a more specific and accurate immunoassay for sepsis diagnosis based on the use of a new panel of markers and optimization of the immunoassay using a novel method for observing the binding kinetics through label free detection. The new methods will be validated by comparison with clinical samples and standards and will also be generally applicable to other clinical diagnostic methods based on colorimetric immunoassays.
CE-microtiter plates and microplate readers:
The CE-microArray project will develop cavity enhanced microtiter plates and microplate readers. These will allow at least a fivefold increase in sensitivity over conventional colorimetric plate readers.
- The simplest device will consist of custom 96 well microtiter plates with high reflectivity dielectric mirrors deposited on the underside and the coverplate, creating an integral optical cavity. This arrangement will allow the microtiter plates to be used in conventional plate readers.
- A second development will integrate dielectric cavity mirrors into the optical path of a conventional colorimetric microplate reader. This will allow the device to be used with standard microtiter plates and result in an improvement in sensitivity of between 30 and 50 fold.
- The final device will combines the integrated cavity mirrors with an array detector to produce a cavity enhanced imaging microplate reader, capable of making simultaneous colorimetric measurements on a glass microarray with a sensitivity enhancement of greater than 5 fold.
Sepsis immunoassay development and optimization:
The CE-microArray project will develop and optimize a novel marker panel for the diagnosis of sepsis.
[1] M. E. Bianchi, Journal of Leukocyte Biology, 81(1), 1 (2007)
[2] R. Daniels, J. Antimicrob Chemother, 66(2), 11 (2011) and references within
[3] Angus DC, Linde-Zwirble WT, Lidicker J, et al: Epidemiology of severe sepsis in the United States: Analysis of incidence, outcome, and associated costs of care. Crit Care Med, 29, 1303 (2001)
[4] J. Soong, and N. Soni, "Sepsis: recognition and treatment." Clinical medicine 12 (3) 276 (2012)
[5] C. Pierrakos, Critical Care, 14(1) 15 (2010)
[6] P. Angenendt, Drug Discovery Today, 10(7), 503-511, (2005)
[7] PricewaterhouseCoopers clinical diagnostics market report (www.pwc.com/diagnostics2011)
Project Results:
CE-microArray was a two-year programme (extended by a total of eight months via a five-month suspension and three-month extension period) which was structured around technological developments related to cavity enhanced microplate readers and the integration of these to create novel prototype cavity enhanced microplate readers which would then be validated using a novel bioassay for sepsis risk detection.
Within the CE-microArray project the work was divided into 7 workpackages (WP). This comprised four RTD workpackages one for validation, one for dissemination and exploitation, and one for project management. The results from the science and technology workpackages (WP 1-5) are described below. Full details of the deliverables are provided in the submitted reports for the individual deliverables.
WP 1 Cavity enhanced microtiter plates
WP1 involved the design and production of novel cavity enhanced microtiter plates and required the identification of polymer microtiter plates suitable for dielectric mirror deposition followed by mirror deposition. There were 2 deliverables associated with this workpackage.
D1.1: Polymer microtiter plate with fractional optical losses of <0.02 per pass
D1.1 aimed to identify high optical quality injection moulded polymer microtiter plates with fractional optical losses of <0.02 per pass.
Polystyrene Falcon® and Nunc™ glass bottom microtiter plates were identified as standard high quality plates with fractional optical losses of < 0.02 (<67%). The glass bottomed microtiter plates, introduced a loss of ~ 8.5% compared to the ~ 52% loss of the lower optical quality polystyrene microtiter plates at 450 nm that is the wavelength of interest for our bioanalytical measurements.
The plates were sent for e-beam deposition and successfully accepted the dielectric coating by using only ambient temperature (without ion assistance – non-IAD). Using a non IAD deposition will lead to reduced process temperature and lower manufacture cost.
D1.2: Microtiter plates with dielectric mirrors deposited on the bottom and on the coverplate
Polystyrene Falcon and Nunc glass bottom microtiter plates were identified as standard high quality plates with fractional optical losses of < 0.02 (<67%) for D1.2. The plates underwent further tests for biochemistry and thermal stress to investigate their suitability for the coating process. Using only the ambient temperature e-beam thermal evaporation technique by YPT the plates successfully accepted the dielectric coating and had a bandwidth around 100 nm centred at either ~450 nm or ~550 nm. Further measurements by TEES showed that the deposited mirror reflectivity was close to 99% and exhibited relatively low optical absorption. The technique appears to be suitable for polymeric materials, it shows reduced process temperature, lower manufacture cost and simpler deposition.
The results on the cavity enhanced plates produced a ~5 fold improvement in sensitivity which is significantly better than a conventional single pass measurement. Further investigation on the alignment and optical quality of plates and covers can in principle lead to a ~10 fold enhancement over commercial colorimetric plate readers.
WP 2 Bioassay implementation and microarray
WP 2 involved the design of bioassays to provide both a more accurate test for sepsis diagnosis and also a means of testing the performance of the cavity enhanced devices produced in WP3 and WP4. There were 4 deliverables associated with this workpackage.
D2.1 Selection of panel of markers
Identifying biomarkers for the diagnosis of sepsis has been subject to discussion and disagreement in recent years. Distinguishing between sepsis and non-infectious inflammation remains a key challenge, and there are hundreds of discussed potential biomarkers for sepsis.
However, as the CE-microArray project intends to benchmark with existing systems, commercialized “standard markers” are aimed for. A standard device for immune diagnostic at University Clinics Ulm, and against which the CE-microArray devices would be compared for accuracy and sensitivity, is the Siemens IMMULITE. D2.1 therefore represented the first step in the development of the devices and their eventual validation.
After consulting and in accordance with Prof. Schneider at University Clinics Ulm, eight markers were chosen as markers for CE-microArray. They cover a large sepsis panel in terms of functionality and are applied at University Clinics Ulm for sepsis diagnostics. These markers also cover several assay challenges as the thresholds of healthy state vary from some pg/ml (IL-1β, IL-6, IL-10, TNF-α) over several tenth pg/ml (IL-8) to µg/ml (LBP). Enzymatically active markers are also included from mU/ml (EPO) to kU/ml (sCD25).
D2.2 Preparation of reference materials
The aim of the deliverable was the preparation of calibrator solutions mimicking an HRP-ELISA reaction. There were three levels of calibrator solutions planned:
a. Static calibrators: mimic an endpoint measurement and are a dye solution
b. Dynamic calibrators: mimic the staining reaction and are a two compound system containing an HRP-active part and a dye part
c. Dynamic handling calibrators: mimic the last step of an ELISA and provide binding moieties to the surface, followed by a one-step binding of a HRP active compound. In accordance with the aims, several commercial easy available dyes were tested for application as static calibrators. The dynamic calibrators were based on streptavidin-HRP and TMB. The dynamic handling calibrators had in addition biotinylated BSA.
The calibrator solutions will enable the partners to run their own measurements independent of the status of the sepsis marker panel. This will decouple the workflow and allow each partner to succeed independent of the results from any other. As the calibrators are benchmarked to IL8 each partner can measure the sensitivity of the according system by also applying it to any other microplate reader capable to read wavelength at 480 or 600 nm.
D2.3 Validation of panel of markers in microplate format
The aim of the deliverable was validation of a panel of markers in microplate format and provision of calibration solutions to project partners.
In accordance with D2.2 a total of 8 biomarkers were selected and according antibodies will be tested. In terms of cost and availability it is in general more cost-efficient to buy several antibodies and test them for compatibility with ELISA, but the success rate can be quite small and the smaller price comes in terms of cost in time as it can take several month to improve a binding pair to diagnostic sensitivity. In a first attempt this was applied to the marker IL8, but it became clear that for the other markers it is more time-saving to simply order the according markers, even if the number of measurements is then drastically limited within the process. In addition it is not possible with such kits to measure the binding kinetics via iRIF as the amount of material on these kits is so small, that an iRIF-measurement would cost around 1,500 EUR per run. Therefore we made for the validation of the IL8 and some other panel markers iRIF-measurements to derive some protocol improvements. As we later applied the complete sets, we could not use the iRIF-setup anymore and simply used the vendor protocols for sake of high sensitivity. From the previous selected 8 biomarkers one failed completely, but the other 7 could be completely established in the lab for measurements in a microplate.
Therefore the deliverable D2.3 was met in all terms, that a full validation in a microplate format was shown and is compatible with the CE-microArray reader. For the purpose of the compatibility Dr. Zuzana Bajuszova from Teesside University visited Freiburg to make jointly the HRP-reaction for detection as well as microcontact printing of structures. This knowhow transfer ensures the compatibility from MTP to the CE-microarray reader.
With this step the validation of the CE-microArray devices against pre-characterized clinical samples in D5.3 can be realized, as now the antibodies and protocols are available for the testing.
D2.4: Testing of chemical protocol for antibody immobilization
D2.4 aimed to establish assay protocols for the biomarkers identified in D2.2. In D2.2 a total of 8 biomarkers were selected and validated in D2.3 accordingly.
It was possible to show how to optimize an assay system applied to the marker IL8, but it became clear that for the other markers it is more time-saving to simply order the according markers, even if the number of measurements is then drastically limited within the process. In addition it is not possible with such kits to measure the binding kinetics via iRIF as the amount of material on these kits is so small, that an iRIF-measurement would cost around 1,500 EUR per run. Therefore we made for the validation of the IL8 and some other panel markers iRIF-measurements to derive some protocol improvements. As we later applied the complete sets, we could no longer use the iRIF set-up and simply used the vendor protocols for the sake of high sensitivity. From the previous selected 8 biomarkers one failed completely, but the other seven could be completely established in the lab for measurements in a microplate.
WP 3 Prototype cavity enhanced microplate reader platform
The overall goal of WP3 was to develop two novel prototype cavity enhanced microplate readers which would be used to make cavity enhanced measurements on standard 96 well microtiter plates and also microarrays. It consisted of three deliverables.
D3.1: A prototype cavity enhanced microplate reader
The deliverable seeks to develop a prototype cavity enhanced microplate reader capable of measurements on single wells of a 96 well micotiter plate.
A fibre-coupled vertical optical cavity was developed and further integrated with a motorized XY stage to form the backbone of the commercial grade instrument in D4.1. The stage functioned as a receptacle for the microtiter plate and allowed cavity enhanced point measurements on individual wells. Fibre-coupling provided the advantage of a relatively easy to align optical setup with well collimated light beam and simple setup geometry.
Task 3.1 also involved the investigation of the effect of the meniscus and the optical quality of the microtiter plates on the optical losses. To minimise losses due to liquid/air interface two approaches were implemented, involving either dosing exact amount of sample fluid for full well capacity or using high quality coverplates covering the surface of sample wells. While accurate filling is likely to be the simplest approach, coverplates have the advantage of ensuring a stable flat fluid interface.
Polystyrene Falcon® and Nunc™ glass bottom microtiter plates were identified as standard high quality plates with fractional optical losses of < 0.02 in D1.1. Their optical quality as part of Task 3.1 was investigated by recording the introduced cavity losses and obtaining the values of cavity enhancement. The insertion of the glass bottomed plate into the cavity with a blank solvent solution provided a loss of ~ 48%. The relative loss by the polystyrene plate due to lower optical quality was ~ 60%. The presence of a coverplate introduced an additional ~ 6% loss. This resulted in a cavity enhancement value of 37 for glass and 23 for polymer bottom plates. The enhancement increased to 58 fold and 41 fold respectively with the method of accurate filling.
The results produced substantial improvement over conventional measurements and met D3.1 aiming to produce a prototype CE-microplate reader with a cavity enhancement factor of > 30. A schematic of D3.1 is shown as figure 1.
D3.2: A prototype cavity enhanced imaging microplate reader capable of multiplexed detection
Originally, the aim of D3.2 was to develop a cavity enhanced imaging plate reader using a point by point scanning in raster scan mode. This would have involved moving the motorised XY stage over small area and simultaneously recording the spectra. However as the developmental work progressed we realised that this approach of acquiring a cavity enhanced image would be relatively time consuming and not the best use of our resources. Therefore the consortium decided to propose a change in the deliverable and produce and interim multiplex prototype for D3.2 which would be further developed in D3.3.
The prototype constructed was based on a 2×2 array of individually assembled dielectric mirrors, collimators and multi-furcated optic fibres. The output was monitored with a multichannel spectrograph allowing two well to be monitored simultaneously.
The experimental work involved obtaining the absorption spectra of two independent set of measurements at the same time, using a single light source, an array of mirrors, a microtiter plate and a single detector. Results showed a CEF value of 30 for channel 1 and a value of 29 for channel 2. The agreement within the values indicates a well aligned array of optical cavities and high accuracy in manufacturing.
Overall, we can conclude that the construction of the intermediate prototype for multiplexing was successful and will be the foundation of the multiplex cavity enhanced plate reader using an imaging spectrometer in D3.3
D3.3: A prototype cavity enhanced imaging microplate reader using a 2D array detector
D3.3 sought to develop a prototype cavity enhanced imaging microplate reader by incorporating a camera into the cavity enhanced optical setup, hence providing a recorded cavity enhanced image with at least 30 fold improvement compared to conventional imaging plate readers.
A free-space coupled CE-imaging plate reader was developed, where a Point Grey monochrome camera operated in 16-bit mode was used to record the images. To identify the most compatible arrangement with imaging, we decided to investigate three cavity configuration. For demonstration purposes and ease of mounting the array was tested on optically clear microscope slides. Key scientific and technical challenges existed in constructing this prototype as it represented a truly novel area with little previous work. During the course of development we established the experimental methodology to obtaining such images and processing them to provide a valid end result. The CEF value contained contributions from surface related scattering losses and was a measure of the total cavity loss. The slides contributed in average 14% loss to the system and were held in a custom made slide holder.
The first optical cavity was formed by two plane mirrors with average reflectivity of 94.5%.This configuration is highly unstable and required a well aligned optical cavity with a well collimated light beam incident on the output mirror. To increase the stability of the system the mirrors were separated by the minimum distance available. The recorded cavity images yielded to a significant colour enhancement and provided 11 fold improvement when compared to conventional imaging. This geometry had no focusing point hence provided a relatively large beam area of ~5 mm in diameter enable to contain a 2×2 array.
The second configuration was formed by one plane and one concave mirror of average reflectivity of 99%. This arrangement is relatively stable but due to the presence of a focusing mirror provided a relatively small centre area of 1 mm in diameter available for imaging. This resulted in reducing the size of the manually spotted array. The geometry also required a well-positioned array through the centre line of the beam to avoid side reflections. The results showed a 27 fold enhancement that was further improved to 43.
The final configuration was the most stable from all three tested, containing two confocal mirrors of 99% reflectivity. Similar to the previous arrangement the presence of the focusing mirror yielded a centre hot spot, requiring a well-positioned array at the centre line of the beam. This introduced additional challenges to the alignment procedure. The resulting cavity enhanced images yielded to a 30 fold improvement when compared to conventional single-pass imaging.
Overall we can conclude that the construction of a CE-imaging prototype was successful and D3.3's proposal of an enhancement of >30 was met. The intention was to proceed with a plane-parallel configuration for D4.2 as it provides a large area for the array, a simpler image processing methodology and has the potential to provide a cavity image with >30 fold enhancement by the use of custom made 99% mirror set.
WP 4 Integration of reader with bioassay
The main aim of WP 4 was to take the prototype lab based cavity enhanced microplate readers developed in WP 3 and engineer them into commercial grade cavity enhanced microplate readers. Some of the bioassays developed in WP 2 were also tested and optimised for use in the microplate readers in this workpackage. Unexpected technical challenges arising for the construction of the prototypes for D4.1 and D4.2 led to a project suspension of five months and a project extension of three months to allow the deliverables to be successfully completed. WP 4 consisted of three deliverables.
D4.1: A commercial grade cavity enhanced microplate reader
The aim of D4.1 was to convert the prototype cavity enhanced microplate reader capable of measurements on single wells of a 96 well microtiter plate, developed in D3.1 into a commercial grade instrument. This work was carried out by consortium partners EII in collaboration with TEES. A stand alone cavity enhanced microplate reader was developed along with the custom software required to operate the instrument and analyse the recorded spectra. A render of the instrument is shown in figure 2. Full details of the mechanical operation of the instrument and the functionality of the software can be found in the submitted report for D4.1.
D4.2: A commercial grade cavity enhanced imaging microplate reader
The aim of D4.2 was to convert the prototype cavity enhanced imaging microplate reader capable of imaging measurements, developed in D3.3 into a commercial grade instrument. This work was carried out by consortium partners EII in collaboration with TEES. A stand alone cavity enhanced imaging microplate reader was developed along with the custom software required to operate the instrument and analyse the recorded spectra. A render of the instrument is shown in figure 3. Full details of the mechanical operation of the instrument and the functionality of the software can be found in the submitted report for D4.2.
D4.3: Tested and improved assay protocols and calibration samples to benchmark readers
The aim of D4.3 was to develop calibration samples and improved assay protocols (ALU-FR) which would be used later on in D5.2 to test the performance of the cavity enhanced plate readers developed in D4.1 and D4.2.
As such ALU-FR provided the knowledge and handling of the standard microarray readers and developed a bBSA as well as a HRP binding assay enabling comparison between the microplate readers used in ALU-FR – a victor reader V3, Berthold technologies, Germany – and the CE microplate readers later on in D5.2.
WP 5 Validation
WP5 aimed to validate the cavity enhanced devices produced by the CE-microArray project. Some of the validation work undertaken within this workpackage was subcontracted as per the DoW to University Clinics Ulm (UCU) (Germany) headed by Prof Marion Schneider as they have great expertise in the diagnosis and treatment of sepsis and related infections. There were four deliverables associated with this workpackage.
D5.1: Ethical clearance
Ethical clearance was obtained the Ethics Commission of the University of Ulm to use anonymised patient data from ongoing clinical studies with intensive care patients as a platform to perform the project’s validation work. An ongoing study was extended, and confirmation was provided by the University of Ulm that the tests wwill use samples from those who have given consent for their samples to be used for the purposes of commercial test development.
D5.2: Evaluation report comparing CE-microArray devices with standard microplate assays
D5.2 sought to validate the CE-microArray devices against calibration samples. Successful application was made of the D4.1 CE-microArray 96 well Plate Reader to standard colorimetric assays and the D4.2 CE-microArray Imaging Plate Reader to bio printed arrays.
For the first test series, a standard STREP-HRP colorimetric assay and a standard Osteocalcin ELISA assay was performed, and the signal at 450 nm was read on the CE-microArray 96 well plate reader. The results were benchmarked against a standard microplate reader and showed 77-91 fold improvement in sensitivity.
In the next test, the CE-microArray Imaging Plate Reader was tested against a microcontact printed bio array (assay developed by ALU-FR) on a glass surface and the results were benchmarked against conventional images taken by the reader. The results showed 6-8 fold enhancement compared to the images taken without the presence of the cavity mirrors.
We can conclude that the validation of the CE-microArray devices against standard colorimetric assay were successful and the proposed enhancement of >30 for prototype D4.1 and enhancement >5 for prototype D4.2 was met.
The final step was to involve the validation of the CE-microArray devices against pre-characterized clinical samples in D5.3.
D5.3: Evaluation report comparing with clinical samples
The aim of D5.3 was to perform comparative measurements on clinical samples using the cavity enhanced microplate readers and a standard microplate reader. This required making the measurements at University Clinics Ulm (UCU) (Germany) under the supervision of Prof Marion Schneider as UCU has the facilities to safely make measurements on clinical samples. Dr Günter Roth from ALU-FR travelled to Ulm with test calibration assays. The cavity enhanced microplate reader was sent by TEES and Dr Zuzana Bajuszova (TEES) travelled to Ulm to make the measurements with the cavity enhanced microplate reader. Unfortunately when the cavity enhanced microplate reader was unpacked in Ulm it was found to have been damaged in transit and was inoperable. This led to the planned comparative measurements having to be cancelled. Repair of the instrument was not possible onsite, would require the visit of engineers from EII and would require several weeks to achieve. As these measurements were planned for the end of the project there was insufficient time remaining to reschedule the measurements. However, from the measurements performed for D5.2 it is possible to estimate that the cavity enhanced microplate readers would be approximately 30 times more sensitive for the D4.1 prototype and 6-8 more sensitive for the D4.2 prototype.
Technicians at Ulm subsequently made some repairs to D4.1 and it will shortly be returned to the UK for realignment. The consortium partners were updated of this situation, and confirmation was received from SME partners ANX, ASYS, BMT, NEB and YPT of their ongoing interest to continue to work together to develop the project results despite this setback (the SMEs are due to form the Joint Venture Board to after the project ends). This is felt to reflect the longer-term value of the project work and its results.
D5.4: Establishing an ex-vivo whole blood stimulation system
To understand the nature of immune dysfunction in patients with sepsis manifesting after severe trauma, a robust whole blood ex vivo test system (using TruCultureTM) was developed as D5.4.
One millilitre of blood was drawn into separate syringes containing ligands to stimulate the following TLR2-9. Amongst many others, the biomarkers TNF-α, Interleukin-1β and TNF-α followed by IL-1RA and soluble TNF-RII was quantified by multiplexed sandwich immunoassays. The IL-1ratio and TNFratio following stimulation with LPS, were defined as the ratios of IL-1β [pg/ml]/IL-1RA [pg/ml] and TNF-α [pg/ml] /sTNF-RII [pg/ml], respectively.
When compared with healthy donors, most TLR induced cytokines were lower in trauma and even lower in sepsis patients’ cultures. An exception was the TLR2 stimulation, which induced more inflammatory and anti-inflammatory cytokines as well as soluble receptors in trauma and sepsis than in healthy donors. Among the other TLR responses, TLR3 was most dramatically downregulated in patients with trauma and even more in sepsis patients. Calculating the IL-1ratio and the TNFratio, we found a patient-type specific ratio of ligand to antagonists. Healthy donors had a median IL-1ratio of 1.48 and a median TNFratio of 2.73 trauma patients had 10times -, and sepsis patients had 100times lower IL-1 and TNFratios.
The developed TruCultureTM ex-vivo whole blood TLR stimulation test is valid to correlate a defined response pattern to the clinically established stages of trauma and sepsis. Results may substantially contribute to signalling pathways leading to immune dysfunction. To link to WP2, the biomarkers identified within WP2 were measured in D5.4.
Potential Impact:
Potential Impact:
The potential impact of the work is based on the further development and commercialisation of the project results:
Result 1, Microtiter plates with dielectric mirrors deposited on the bottom and on the coverplate, which have the potential to be commercialised as a consumable which gives significantly improved sensitivity compared to a normal plate.
Result 2, Tested chemical protocol antibody immobilization and assay protocol, which will require further research, development and validation, leading to further testing and clinical trials. This is expected to take at least a further two years.
Results 3 and 4, prototype and commercial-grade cavity enhanced imaging microplate readers, and the Cavity Enhanced Microplate Reader – these will be positioned as highly sensitive microplate readers.
Result 5, Establishing an ex-vivo whole blood stimulation system, which will link to result 2 and will contribute to the aim of developing diagnostic tests for sepsis.
The main focus is on the commercial impact which will result from further developing the results and taking them to market, and also the wider societal implications which would result.
In terms of economic impact, the successful commercialisation of the results would result in increased sales, turnover and potential job creation within the consortium companies.
In terms of broader societal impact, the ultimate aim of the project is to develop a more sensitive, accurate, faster and more useful diagnostic platform for sepsis, and to help to provide patients with early, accurate diagnosis. Sepsis is the most prevalent cause of death in non-coronary Intensive Care Units (ICUs), and the mortality rate in patients admitted to hospital with severe sepsis is 28-50%. The potential impact of the project in helping to address this situation is therefore substantial, in terms of improving mortality rates, supporting healthcare efficiency and reducing costs. It has been estimated that in the US ~$17 billion is spent annually treating sepsis in patients and that a similar amount is spent across the EU (Angus et al, 2001).
Patent searches have been undertaken as part of the preparation of the interim and final Plans for the Use and Dissemination of Knowledge, and nothing has been found which would restrict the consortium’s freedom to operate and to commercialise / exploit the project results.
As explained in several Deliverable reports including the Final Plan for the Use and Dissemination of Knowledge, it will take an estimated two more years for additional testing and clinical trials of result two (the tested chemical protocol antibody immobilization and assay protocol) as part of the integration of the project results into a diagnostic platform. In the shorter-term, impact will be realised from the further development and progress towards commercialisation of results one, three and four.
Result one has the potential to be commercialised as a consumable which offers increased sensitivity compared to standard plates.
For the Cavity Enhanced Microplate Reader and the Cavity Enhanced Imaging Microplate Reader, the partners will target more broadly the microplate reader market, including as medical diagnostic equipment. The key decision factor when purchasing a new plate reader is sensitivity, and there is a large market for low cost, highly sensitive absorbance based microplate readers. D6.2 the Market Report, showed that the annual global market for microplate readers was estimated to $300 million according to the survey in 2009 (Banks, 2009).
D6.2 also outlines that the lifetime of the proposed reader will be at least five years, based on the relatively long expected lifetime of its individual components. By providing an increased sensitivity over standard absorption microplate readers at the same price point, the consortium aims to achieve a 1% market share in the single mode absorbance microplate reader and absorbance based imaging microplate reader markets.
D4.1 concludes that the validation of the CE-microArray devices against standard colorimetric assay was successful, and that the proposed enhancement of greater than 30-fold for prototype D4.1 and greater than 5-fold for prototype D4.2 was met. This provides a strong basis for the realisation of the project’s impact potential.
Main Dissemination Activities:
The main dissemination activities which have been carried out so far are as follows.
Activity aimed at an industrial or end-user audience:
- ASYS showcased the project results at the PHOTONEX 2016 exhibition in Coventry, UK, on 12th and 13th October 2016. This is a trade show for photonics and light-based technologies, with an audience of industrial representatives from the UK and overseas.
- The promotional pamphlet developed during the project as D6.6 was printed and taken to the PHOTONEX exhibition to be handed to delegates.
- TEES was one of 23 organisations which took part in a visit to India in May / June 2016. This was led by the Chairman of NHS England and aimed to showcase research and innovation with healthcare applications, and to develop collaborative links. CE-microArray was part of the research showcased by TEES during this visit.
- A promotional video (duration 1 minute, 53 seconds) was produced as part of the visit to India to provide information on the aims and work of CE-microArray. This shows prototype D4.1 being used in the TEES laboratories.
- A press release, available at
http://www.tees.ac.uk/sections/research/news_story.cfm?story_id=4608&this_issue_title=March%202014&this_issue=250, was issued by Teesside University at the start of the project and subsequently formed the basis of articles about CE-microArray in, amongst others, LabMate Online (based in the UK), Nursing Standard (UK), Pharmiweb.com (UK), Pharma Focus Asia (India), PR Newswire UK, Wallstreet: Online (Germany), Informazione.it (Italy), and local newspapers and radio stations in Middlesbrough and the north-east of England.
- An entry for the project was created on the research section of the Teesside University, ALU-FR and EII websites respectively, alongside overviews of other projects and activity.
The website was updated during the project and a total of 19,625 hits from 4,295 visits were recorded over the project lifetime (to 23rd November 2016) by the project website platform, iPage. The Deliverables which were identified at the start of the project as ‘Public’ (i.e. not commerciality sensitive or confidential) have been made available on the website. These are the promotional leaflet (D6.6) and the report on regulatory and standardisation requirements in EU and US markets (D6.5).
The main dissemination activities to a scientific audience are:
Two journal papers were published:
- Bajuszova, Z., Ali, Z., Scott, S., Seetohul, L. N., Islam, M. (2016) 'Cavity Enhanced Immunoassay Measurements in Microtiter Plates using BBCEAS' Analytical Chemistry; 88 (10): 5264-5270, DOI 10.1021/acs.analchem.6b00375.
- Burger, J., Rath, C., Wöhrle, J., Meyer, P.A. Anmar, N.B. Kilb, N., Brandstetter, T., Pröll, F., Proll, G. Urban, G., Roth, G. (2016), ‘Low-Volume Label-Free Detection of Molecule-Protein Interactions on Microarrays by Imaging Reflectometric Interferometry’ Journal of Laboratory Automation, DOI 10.1177/2211068216657512.
One conference presentation was given by the project coordinator, Prof. Meez Islam, who presented the project during a talk on ‘Novel Applications of Liquid-phase CE Absorption Spectroscopy’ at the Cavity Enhanced Spectroscopy (CES) conference, which was held in Boulder, Colorado, USA, June 16th – 19th 2015.
Further dissemination activity is planned as follows for the initial period after the project:
Journal articles:
- A journal article on the development of result 1, the microtiter plates with dielectric mirrors deposited on the bottom and on the coverplate, and the increase in sensitivity achieved with the plates. This will be co-authored by TEES and YPT.
The target journal for this paper is Analyst, which is published by the Royal Society of Chemistry in the UK and supports Open Access publishing via either Gold or Green routes (after a 12-month embargo period in the case of Green Open Access).
- Two journal articles on cavity enhanced imaging, the preparation of which will be led by TEES. The target journals are Optics Express, which is published by the Optical Society of America and also support Open Access via Gold or Green routes (with a 12-month embargo period for Green Open Access); and PLOS ONE, which is published by the non-profit published PLOS, and which supports Open Access publishing with no embargo on Green Open Access.
Exhibitions / trade shows:
- ASYS will follow-up from contacts made at the PHOTONEX exhibition in October 2016 – several companies expressed an interest in the project devices, and it would be beneficial to develop these contacts further (although as stated in section 3.1 further development work will be needed before commercialisation can proceed).
- The potential of attending European trade shows will be explored, notably MEDICA, the World Forum for Medicine, which takes place in Düsseldorf in Germany. Due to the respective stages of development of the project results, and the need for further development work of results two and five, the initial focus would be on the Cavity Enhanced Microplate, the Cavity Enhanced Microplate Reader and the Cavity Enhanced Imaging Microplate Reader. Partners will also carry out an overview of other relevant trade shows to identify possible targets as the development of the project results progresses.
The project website will also be maintained following the end of the project.
A significant focus of dissemination activity in the period immediately following the end of the project will be to build links with other companies whilst continuing to develop the project results. Partners’ current contacts and networks will be utilised to raise awareness of the results, and opportunities will be taken to meet company representatives face-to-face. It is anticipated that this may lead to the development of collaborative activity, including the development of additional applications for external funding to further develop the project results.
Exploitation of Results:
The aim is to develop the following three products:
1. Cavity Enhanced Microplate (result 1)
A modified microplate with integral dielectric mirrors to enhance the operation of microplate readers.
2. Cavity Enhanced Microplate Reader
A diagnostic tool made to perform cavity enhanced absorption measurements on a standard 96 microwell array.
3. Cavity Enhanced Imaging Microplate Reader (result 3, 4)
A more complex diagnostic tool incorporating a 2D detector capable of making multiplex imaging measurements on 96 well microtitre plates.
The aim is also to further develop result 2, a tested chemical protocol antibody immobilization and assay protocol, result 5, and an ex-vivo whole blood stimulation system.
As stated above, the overall objective is to combine these products to address the ultimate goal of developing a more sensitive, accurate, faster and more useful platform for the diagnosis of sepsis. As results 2 and 5 are expected to take a longer time to develop, emphasis will be placed in the intervening period on developing the Cavity Enhanced Microplate, the Cavity Enhanced Microplate Reader and the Cavity Enhanced Imaging Microplate Reader for other markets as they are closer to commercialisation, whilst maintaining a focus on the overall goal.
The Final Plan for the Use and Dissemination of Knowledge sets out pathways towards the commercialisation and exploitation of the results. These are as follows:
Result 1:
This result is to be positioned as a consumable. This project result will require further development work, as the process which has been developed within the project to deposit the mirrors is relatively expensive. A lower-cost process would be required to position this as a competitively-priced consumable. The current process also takes a relatively long time, and is limited to the production of batches of plates, rather than a more advantageous continuous process.
In terms of advantages, this project result has the potential to fill a significant gap in the market, as in the partners’ considerable experience of this market, no similar plate is currently available. Initial measurements collected as part of the project indicate that the plates give significantly improved sensitivity compared to a normal plate, which has the potential to be appealing within the marketplace.
This project result would be marketed as a disposable consumable, so has the potential to result in a strong revenue stream.
Further funding is needed to explore and optimise the process of producing the plates. This would take more time and concentrated effort than has been available in CE-microArray.
In the longer-term, a partnership may be required with a large manufacturer to make the plates on a sufficiently large scale.
Prospective lead partners: YPT, UP, working with ASYS
Result 2:
As stated in D6.4 further research, development and validation will be required after the project to further develop result 2, leading to further testing and clinical trials. This is expected to take at least a further two years. This is particularly the case as project resulted in a smaller number of antibody pairs than was planned at the start of CE-microArray.
Also as stated in D6.4 result two has led to the additional benefit of supporting BMT in extending their iRIfS technology and its uses and applications.
Further funding and investment will be necessary, targeting national or European funding at first to make more progress before using the results to target commercialisation via large clinical diagnostics companies.
The partners have some contacts with these companies, which would add support to funding applications and the later activity.
Prospective lead partners: BMT, NEB, with the support of ASYS
Result 3 and 4:
The aim is to position results 3 and 4, and the Cavity Enhanced Microplate Reader, to meet a market need for highly sensitive microplate readers.
Further development work will be needed after the end of the project – as stated in D7.5 some of the devices’ current components are relatively expensive, and an initial focus will be to reduce the cost by simplifying their construction whilst safeguarding / improving usability.
The aim will be to build links with companies in parallel to this activity in order to demonstrate and raise interest in the results, but it is anticipated that the cost will need to be reduced before the interest of these companies can be secured.
Following this stage, the main options will be:
- for a larger company to purchase the IP and develop the products as part of their own technology / product range. This would rely on finding a company with very strong interest in the results, and it would also end the partners’ ability to further develop them;
- the partners further developing the project results themselves, and to collaborate with a smaller company to sell or distribute them as final products. This would allow the partners to retain greater control and to build their own reputation as the products would carry their branding and become part of their range. This would be a preferable option from the perspective of ASYS as project Exploitation Manager.
More funding will be required to reduce the costs of the prototypes.
Potential targets include the Emerging and Enabling Technologies programme or the Small Business Research Initiative programme, both of which are offered by Innovate UK. The H2020 SME Instrument is another possible target.
In addition, TEES is a partner in the H2020 GateOne project, GA number 644856, which is developing demonstrator technology for SMEs. This may provide opportunities for cross-fertilisation and for the further shared or joint development of the CE-microArray and GateOne products.
Prospective lead partners: ASYS, ANX
Result 5:
This result contributes to the aim of developing diagnostic tests for sepsis, and will be of interest to clinicians. It is based on the use of the existing TruCulture system, so will contribute to the work required to further develop result 2.
The result will be taken forward as part of the development of result 2: further funding will be required for additional clinical testing and validation.
Prospective lead partners: BMT, NEB
In summary, the exploitation of the results has not yet begun in earnest as the focus has been on delivering the technical project work packages and the demonstration work package. In particular, it was not possible to complete Deliverable 6.8 the invitational meeting targeted at producers of diagnostics, as there was simply insufficient time within the project lifespan between the completion of the project results and the end of the project period. It was therefore not possible to undertake advanced dissemination of this type, which would require a longer lead-in time and will more feasibly be carried out after the completion of the project and a period of further development and refinement work. It is felt important that direct dissemination of this kind to a group of companies takes place only when the project devices (D4.1 D4.2) are ready to be presented to and scrutinised by such an audience. It will be vital to take this opportunity to build a strong reputation and ensure as far as possible that a strong impression is made and that good relationships begin to be forged.
In order to gain initial feedback from relevant companies, ASYS showcased project devices D4.1 and D4.2 at the PHOTONEX trade exhibition in Coventry, UK, on 12th and 13th October 2016. This is a trade show for photonics and light-based technologies, with an audience of industrial representatives from the UK and overseas. Although the exhibition took place after the end of the project, the majority of the preparation took place during the project lifetime. Attendance at this exhibition allowed ASYS to make connections with companies who are interested in the project devices, and served as preparation for the more advanced dissemination activity which will take place subsequently.
The results of the technical work packages provide a strong foundation on which to build dissemination and commercialisation activity.
Dissemination activity in work package 6 has therefore mainly focused on preparing for dissemination following the end of the project. This has been done via D6.2 the report on market analysis and potential for microplates and microplate readers; D6.5 the report on regulatory and standardization requirements in EU and US markets; D6.6 the pamphlet for distribution to end-users; and D6.7 the final plan for the use and dissemination of knowledge. These Deliverables will guide dissemination and commercialisation activities following the end of the project.
List of Websites:
Website address: www.cemicroarray.com. Contact details: Professor Meez Islam, m.islam@tees.ac.uk.