Browsing by Author "Tothill, Alexander M."

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    Developing a proof of principle 3D-printed lab-on-a-disc assay platform.
    (2017-05) Tothill, Alexander M.; James, Stephen W.; Partridge, Matthew; Tatam, Ralph P.
    A 3D-printed microfluidic lab-on-a-disc (LOAD) device was designed and manufactured using a low cost (˜£1600) consumer grade fused deposition modelling (FDM) Ultimaker 2+ 3D printer with imbedded microfluidic channels 1 mm wide, 400 μm depth and with a volumetric capacity of approximate 23 μl. FDM printers are not typically used, or are capable, of producing the fine detailed structures required for microfluidic fabrication; in addition 3D-printed objects can suffer from poor optical transparency. However, in this work, imbedded microfluidic channels were produced and the optical transparency of the device was improved though manufacture optimisation to such a point that optical colourimetric assays can be performed in a microfluidic cuvette device with sample path length of 500 μm and volumetric capacity of 190 μl. When acetone vapour treatment was used, it was possible to improve transparency of plastic samples by up to a further 30%. The LOAD device is capable of being spun using an unmodified optical disc drive (ODD), demonstrating the centrifugation based separation of plasma from whole blood in a low-cost FDM 3D-printed microfluidic LOAD device. A cholesterol assay and glucose assay was developed and optimised using cholesterol oxidase (ChOx) or glucose oxidase (GlOx) respectively and horseradish peroxidase (HRP) for the oxidative coupling of chromotropic acid (CTA) and 4-aminoantipyrine (AAP). This produced a blue quinoneimine dye with a broad absorbance peaking at 590 nm for the quantification of cholesterol/glucose in solution. The colourimetric enzymatic cascade assays were developed for use within low-cost FDM 3D-printed microfluidic devices to demonstrate the capabilities and functionality of the devices. For comparison, the assay was run in standard 96 well plates with a commercial plate reader. The results demonstrated that the quantification of 0-10 mM glucose solution using a 3D-printed microfluidic optical device had a performance comparable to a plate reader assay; glucose assay in whole blood samples R² = 0.96.
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    Fabrication and optimisation of a fused filament 3D-printed microfluidic platform
    (IOP Publishing: Hybrid Open Access, 2017-02-15) Tothill, Alexander M.; Partridge, Matthew; James, Stephen W.; Tatam, Ralph P.
    A 3D-printed microfluidic device was designed and manufactured using a low cost ($2000) consumer grade fusion deposition modelling (FDM) 3D printer. FDM printers are not typically used, or are capable, of producing the fine detailed structures required for microfluidic fabrication. However, in this work, the optical transparency of the device was improved through manufacture optimisation to such a point that optical colorimetric assays can be performed in a 50 µl device. A colorimetric enzymatic cascade assay was optimised using glucose oxidase and horseradish peroxidase for the oxidative coupling of aminoantipyrine and chromotropic acid to produce a blue quinoneimine dye with a broad absorbance peaking at 590 nm for the quantification of glucose in solution. For comparison the assay was run in standard 96 well plates with a commercial plate reader. The results show the accurate and reproducible quantification of 0–10 mM glucose solution using a 3D-printed microfluidic optical device with performance comparable to that of a plate reader assay.
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    Optical Detection of Glucose Using 3D-Printed Microfluidic Platform. BIOS2016 Gothenburg
    (Cranfield University, 2016-05-26 11:28) Tothill, Alexander M.
    Poster and data presented at Biosensors 2016 Conference Gothenburg Sweden.
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    Ultrasensitive detection of endotoxins using computationally designed nanoMIPs
    (Elsevier, 2016-06-21) Altintas, Zeynep; Abdin, Mohammed J.; Tothill, Alexander M.; Karim, Kal; Tothill, Ibtisam E.
    Novel molecularly imprinted polymer nanoparticles (nanoMIPs) were designed for endotoxin from Escherichia coli 0111:B4, using computational modeling. The screening process based on binding energy between endotoxin and each monomer was performed with 21 commonly used monomers, resulting in the selection of itaconic acid, methacrylic acid and acrylamide as functional monomers due to their strong binding interaction with the endotoxin template. The nanoMIPs were successfully synthesized with functional groups on the outer surface to aid in the immobilization onto sensor surface. The solid phase photopolymerization approach used for the synthesis of nanoMIPs ranging from 200 to 235 nm in diameter. The limit of detection and KD were significantly improved when endotoxin samples were prepared using a novel triethylamine method. This improved the efficiency of gold nanoparticle functionalization by targeting the subunits of the endotoxin. Compared to the vancomycin MIP control, the endotoxin MIPs displayed outstanding affinity and selectivity towards the endotoxin with KD values in the range of 4.4–5.3 × 10−10 M, with limits of detection of 0.44 ± 0.02 ng mL−1 as determined by surface plasmon resonance (SPR) sensor when itaconic acid was used as the functional monomer. The MIP surface can be regenerated >30 times without significant loss of binding activity making this approach highly cost effective for expensive analyte templates. The combination of molecular modeling and solid phase synthesis enabled the successful synthesis of nanoMIPs capable of recognition and ultrasensitive detection of endotoxins using the highly sensitive SPR biosensor with triethylamine method.