Title: Rapid detection of infectious agents at point of triage (PoT).
Funder: Department of Health (England)

Abstract: WP1. Assays for small volumes and low target numbers (UoS and HPA) . Reduction of sample volume to sub uL volume requires increased sensitivity. A typical immunoassay has a LoD in the pM range - only a few thousand molecules in a 500nL droplet. We will develop enhanced amplification methods to extend the LoD whilst keeping the assay time under one hour. Optimal strategies for assay readout need to be explored, as will time for assay and dynamic range. The HPA has developed many optimised assays which are directly relevant to clinical diagnosis of ESBLs in urinary tract and bloodstream infections and in haemolytic diarrhoea (e.g. the recent outbreak of E.coli 0104). Antibodies will be conjugated to magnetic nano-beads for localisation and washing on-chip. Non-specific binding and loss of target molecules will be addressed through appropriate surface coating strategies. Nucleic acid amplification methods will use solid phase extraction and isothermal techniques developed by HPA. These assays will be optimised to function on the device in sub uL volumes, and the sensitivity of the assay to temperature stability and interfering proteins will be investigated and resolved. WP2. System Hardware (SLE and UoS) (i) Magnetic bead handling methods developed to enable localisation and concentration of the beads for the immunoassays. (ii) Optical readout hardware developed, using micro-optical components. Two different detection systems (fluorescent and chemi-luminescent) will be evaluated. (iii) Integration of droplet position sensors for feedback control of droplet volume and position. Coupled with improved software will improve droplet handling, increasing QC, reducing assay variability and enabling on chip self calibration. (iv) Reagent and sample dispensing techniques will be developed to provide efficient delivery of sample from the “real world” to the chip. 3-D microfluidic components will be designed for buffer and sample pre-processing. (v) User interface, data processing and readout software will be developed to provide a simple interface. This will provide results of the assay in a useful end format while retaining all raw data. (vi) New device designs to be fabricated by Sharp Corporation Japan to improve performance of the device. WP3. Clinical validation (HPA and UoS) (i) Translation of miniaturised nucleic acid assay and immunoassay onto devices. (ii) Testing/validation of assay against accepted methods (ELISA and PCR). (iii) Testing device with spiked real samples. (iv) Processing clinical samples/different matrices. Type strains and commonly occurring contaminant organisms will be spiked into a typical sample matrix to demonstrate sensitivity and specificity. Clinical samples will be accessed through the HPA Regional Microbiology Network and tested in blinded panels. Data will be compared to existing diagnostic tests currently used for diagnosis and/or confirmation. Upstream extraction protocols will be optimised for different clinical matrices, including urine, blood and stool samples, based on established protocols at HPA. Development of sampling handling and preparation platform on the system; Robust protocols for sample loading, dispensing, mixing and washing will be developed that automate conventional methods, but with sub microlitre volumes Milestones: M1 12 months Miniaturised nucleic acid assay M2 18 months Miniaturised Immunoassay M3 18 months On chip Nucleic acid M4 18 months Reproducible droplet volume M5 18 months Miniature optical read-out system developed M6 24 months On chip Immunoassay M7 24 months Complete hardware demonstrating Assay M8 36 months Assays on clinical samples/concordance established M9 39 months Final report


Research activity codes: 4.1 DISCOVERY AND PRECLINICAL TESTING OF MARKERS AND TECHNOLOGIES
Health categories: INFECTION