Final Report Summary - CD-HLASENS (Celiac Disease HLA typing using microfabricated genoSENSarray)
The overall objective of CD-HLAsens is to develop a generic technological platform for point-of-care diagnostics capable of genomic detection with electrochemical transduction. As a model system, biosensor arrays for the screening of coeliac disease will be developed based on the concurrent detection of nucleic acids associated with genetic predisposition, i.e. HLA typing. This overall objective can be subdivided into the following sub-objectives:
• Evaluation of novel sensor surface patterning (nanostructuring, polymer hydrogel)
• Electrochemical detection schemes for the detection of coeliac disease susceptibility associated alleles (low resolution HLA typing)
• On chip integration (microfluidics, temperature control)
• On chip assay development for HLA typing in <15 minutes
• Clinical validation and application of developed biosensor arrays to real blood samples
Such a detection strategy could be used for general population screening as well as for neonatal screening. Together with classical assay formats, novel biomolecule detection schemes based on supramolecular associations will be developed with the aim to fine tune the specificity and sensitivity of the sensors.
Initial work consisted in the identification of the alleles to be detected and the design of the primers to perform the assay. Through the URV contact with the Finish Red Cross, the alleles DQB1 0201, 0202 and 0302 as well as DQA1 0501 and 0505 were selected for the project.
The surface chemistry, inspired from the Fellows’ previous experience in DNA self-assembled monolayer was optimised and allowed reaching very low limits of detection for the targeted DNA was very low current background. The Fellow successfully designed a planar electrode array as well as the accompanying connecting set-up taking full potential of the 64 –channel potentiostat available at the host.
The set-up was supplemented with a temperature control system using Labview and automated using a set of pumps and valves to analyse up to 5 consecutive samples. The Fellow also investigated ways to reduce the cost of the assay and chose to eventually have the electode arrays manufactured using standard PCB manufacturing processes. New surface chemistry based on polymer-DNA conjugates and block copolymer nanostructures were also investigated and the results published. However, the DNA SAM approach was taken forward for the remaining of the project. The fellow modified the contact spotter available at the host to automate the functionalization of the electrode arrays and limit the manual preparative steps needed in order to reduce array-to-array variability.
With the support of the Host’s contacts, a PCR assay was developed that leads to the production of double stranded DNA products with RNA “tails” or “heads” that could be further digested therefore allowing the products to be directly captured at the sensor surface and enzyme labelled for detection.
The assay conditions were further optimised to enable the detection of the PCR product in less than 15 minutes with excellent reproducibility. Finally the system was tested for the multiplexed detection of the selected alleles successfully. The long term stability of the modified sensors was also studied and found to be approximately equivalent to 2 years if stored at 4ºC.
• Evaluation of novel sensor surface patterning (nanostructuring, polymer hydrogel)
• Electrochemical detection schemes for the detection of coeliac disease susceptibility associated alleles (low resolution HLA typing)
• On chip integration (microfluidics, temperature control)
• On chip assay development for HLA typing in <15 minutes
• Clinical validation and application of developed biosensor arrays to real blood samples
Such a detection strategy could be used for general population screening as well as for neonatal screening. Together with classical assay formats, novel biomolecule detection schemes based on supramolecular associations will be developed with the aim to fine tune the specificity and sensitivity of the sensors.
Initial work consisted in the identification of the alleles to be detected and the design of the primers to perform the assay. Through the URV contact with the Finish Red Cross, the alleles DQB1 0201, 0202 and 0302 as well as DQA1 0501 and 0505 were selected for the project.
The surface chemistry, inspired from the Fellows’ previous experience in DNA self-assembled monolayer was optimised and allowed reaching very low limits of detection for the targeted DNA was very low current background. The Fellow successfully designed a planar electrode array as well as the accompanying connecting set-up taking full potential of the 64 –channel potentiostat available at the host.
The set-up was supplemented with a temperature control system using Labview and automated using a set of pumps and valves to analyse up to 5 consecutive samples. The Fellow also investigated ways to reduce the cost of the assay and chose to eventually have the electode arrays manufactured using standard PCB manufacturing processes. New surface chemistry based on polymer-DNA conjugates and block copolymer nanostructures were also investigated and the results published. However, the DNA SAM approach was taken forward for the remaining of the project. The fellow modified the contact spotter available at the host to automate the functionalization of the electrode arrays and limit the manual preparative steps needed in order to reduce array-to-array variability.
With the support of the Host’s contacts, a PCR assay was developed that leads to the production of double stranded DNA products with RNA “tails” or “heads” that could be further digested therefore allowing the products to be directly captured at the sensor surface and enzyme labelled for detection.
The assay conditions were further optimised to enable the detection of the PCR product in less than 15 minutes with excellent reproducibility. Finally the system was tested for the multiplexed detection of the selected alleles successfully. The long term stability of the modified sensors was also studied and found to be approximately equivalent to 2 years if stored at 4ºC.