consortium developing electronic self-testing instruments for sexually transmitted infections usi... more consortium developing electronic self-testing instruments for sexually transmitted infections using nucleic acid amplification testing (NAAT). A proprietary sample collection device has been designed to integrate directly with a microfluidic cartridge. Cell lysis was conducted using a chemical method and nucleic acid purification was done on an activated cellulose membrane. The microfluidic device incorporates passive mixing of the lysis-binding buffers and sample. Preliminary results have shown extraction efficiencies for this new membrane of 69 % and 57 % compared to the commercial Qiagen extraction method of 85 % and 59.4 % for 0.1ng/µL and 100ng/µL salmon sperm DNA spiked in phosphate buffered solution. Preliminary extraction experiments in the passive mixer cartridges with lysis and nucleic acid purification showed extraction efficiency around 80 % of the commercial Qiagen kit. Isothermal amplification was conducted using thermophillic helicase dependant amplification. A low co...
Advances in microfluidics and the introduction of isothermal nucleic acid amplification assays ha... more Advances in microfluidics and the introduction of isothermal nucleic acid amplification assays have resulted in a range of solutions for nucleic acid amplification tests suited for point of care and field use. However, miniaturisation of instrumentation for such assays has not seen such rapid advances and fluorescence based assays still depend on complex, bulky and expensive optics such as fluorescence microscopes, photomultiplier tubes and sensitive lens assemblies. In this work we demonstrate a robust, low cost platform for isothermal nucleic acid amplification on a microfluidic device. Using easily obtainable materials and commercial off-the-shelf components, we show real time fluorescence detection using a low cost photodiode and operational amplifier without need for lenses. Temperature regulation on the device is achieved using a heater fabricated with standard printed circuit board fabrication methods. These facile construction methods allow fabrications at a cost compatible with widespread deployment to resource poor settings.
Algae are considered to be a good source of alternative renewable energy for biofuel as up to 60%... more Algae are considered to be a good source of alternative renewable energy for biofuel as up to 60% of the world is covered in water and contains algae, chlorophyll and plankton, where each of these absorbs light and fixes carbon dioxide through photosynthesis [1]. Wavelength of the light source plays an important role in the algal growth. In this study, the experimental arrangement implemented to study the effects of using blue lasers as coherent light sources on the growth of Algae is explained in comparison to that of the white light LED irradiation. Three individual lasers having the following wavelengths (405 nm, 450 nm and 473 nm) were employed to generate photons with emission spectra matching the pigments' absorption spectra of Chlamydomonas reinhardtii algae; this was with a view to improving transportation of electrons within photosystems II and I, thus expecting an increase in cell division rate. C. reinhardtii algae effectively grew under exposure to both the blue lase...
A method has been developed using chitosan impregnated on membranes reducing the complexity of nu... more A method has been developed using chitosan impregnated on membranes reducing the complexity of nucleic acid extraction. This can be automated in a microfluidic system prior to nucleic acid amplification and detection. The procedure is pH dependent and involves the binding of DNA at pH 5.0 with DNA release at pH 9.0. The results showed extraction efficiencies of 63% and 82% for input samples of 100ng/µL and 0.1ng/µL respectively in comparison to benchtop Qiagen extraction results of 59% and 85%.
Antimicrobial resistance (AMR) is threatening modern medicine. While the primary cost of AMR is p... more Antimicrobial resistance (AMR) is threatening modern medicine. While the primary cost of AMR is paid in the healthcare domain, the agricultural and environmental domains are also reservoirs of resistant microorganisms and hence perpetual sources of AMR infections in humans. Consequently, the World Health Organisation and other international agencies are calling for surveillance of AMR in all three domains to guide intervention and risk reduction strategies. Technologies for detecting AMR that have been developed for healthcare settings are not immediately transferable to environmental and agricultural settings, and limited dialogue between the domains has hampered opportunities for cross-fertilisation to develop modified or new technologies. In this feature, we discuss the limitations of currently available AMR sensing technologies used in the clinic for sensing in other environments, and what is required to overcome these limitations.
A point of care device utilising Lab-on-a-Chip technologies that is applicable for biological pat... more A point of care device utilising Lab-on-a-Chip technologies that is applicable for biological pathogens was designed, fabricated and tested showing sample in to answer out capabilities. The purpose of the design was to develop a cartridge with the capability to perform nucleic acid extraction and purification from a sample using a chitosan membrane at an acidic pH. Waste was stored within the cartridge with the use of sodium polyacrylate to solidify or gelate the sample in a single chamber. Nucleic acid elution was conducted using the RPA amplification reagents (alkaline pH). Passive valves were used to regulate the fluid flow and a multiplexer was designed to distribute the fluid into six microchambers for amplification reactions. Cartridges were produced using soft lithography of silicone from 3D printed moulds, bonded to glass substrates. The isothermal technique, RPA is employed for amplification. This paper shows the results from two separate experiments: the first using the RPA control nucleic acid, the second showing successful amplification from Chlamydia Trachomatis. Endpoint analysis conducted for the RPA analysis was gel electrophoresis that showed 143 base pair DNA was amplified successfully for positive samples whilst negative samples did not show amplification. End point analysis for Chlamydia Trachomatis samples was fluorescence detection that showed successful detection of 1 copy/μL and 10 copies/μL spiked in a MES buffer.
Sustained observations of microbial dynamics are rare, especially in southern hemisphere waters. ... more Sustained observations of microbial dynamics are rare, especially in southern hemisphere waters. The Australian Marine Microbial Biodiversity Initiative (AMMBI) provides methodologically standardized, continental scale, temporal phylogenetic amplicon sequencing data describing Bacteria, Archaea and microbial Eukarya assemblages. Sequence data is linked to extensive physical, biological and chemical oceanographic contextual information. Samples are collected monthly to seasonally from multiple depths at seven sites: Darwin Harbour (Northern Territory), Yongala (Queensland), North Stradbroke Island (Queensland), Port Hacking (New South Wales), Maria Island (Tasmania), Kangaroo Island (South Australia), Rottnest Island (Western Australia). These sites span ~30° of latitude and ~38° longitude, range from tropical to cold temperate zones, and are influenced by both local and globally significant oceanographic and climatic features. All sequence datasets are provided in both raw and proce...
In the standard-care period, 40/41 (97.6%, 95% CI: 87.1-99.9) received treatment; 15 (37.5%) were... more In the standard-care period, 40/41 (97.6%, 95% CI: 87.1-99.9) received treatment; 15 (37.5%) were treated presumptively on the same day (due to symptoms/risk) and 25 (62.5%) were treated on the basis of the laboratory test result; and of these 44% were treated in 1-7 days, 44% in 8-14 days and 12% in 15+ days, with a median time-to-treatment of 8 days. Conclusion This site is already part of a strong and comprehensive STI control program run by NHC over 20 years. Early findings from TTANGO show further improvement in STI management were achieved with point-of-care tests, with treatment occurring on average 8 days sooner for those treated on the basis of a test result. Future analyses will include all 12 clinics and also assess if re-infections have reduced. Disclosure of interest statement No pharmaceutical grants were received in the development of this study. TTANGO is funded by a NHMRC project grant. The GeneXpert cartridges were purchased from Cepheid and Cepheid provided machines on loan for the duration of the study.
This paper presents the design of a modular point of care test platform that integrates a proprie... more This paper presents the design of a modular point of care test platform that integrates a proprietary sample collection device directly with a microfluidic cartridge. Cell lysis, within the cartridge, is conducted using a chemical method and nucleic acid purification is done on an activated cellulose membrane. The microfluidic device incorporates passive mixing of the lysis-binding buffers and sample using a serpentine channel. Results have shown extraction efficiencies for this new membrane of 69% and 57% compared to the commercial Qiagen extraction method of 85% and 59.4% for 0.1ng/µL and 100ng/µL salmon sperm DNA respectively spiked in phosphate buffered solution. Extraction experiments using the serpentine passive mixer cartridges incorporating lysis and nucleic acid purification showed extraction efficiency around 80% of the commercial Qiagen kit. Isothermal amplification was conducted using thermophillic helicase dependant amplification and recombinase polymerase amplification...
training could be improved with requests for a formalised attachment, formal qualification and gr... more training could be improved with requests for a formalised attachment, formal qualification and greater training in practical procedures. The BASHH GD SIG, in liaison with BASHH, aims to optimise GD training for registrars. Plans for improved resources are in progress, including a practical skills course and e-learning.
Brunel DoCLab is part of the esti2 consortium developing electronic self-testing instruments for ... more Brunel DoCLab is part of the esti2 consortium developing electronic self-testing instruments for sexually transmitted infections using nucleic acid amplification testing (NAAT). A proprietary sample collection device has been designed to integrate directly with a microfluidic cartridge. Cell lysis is conducted using a chemical method and nucleic acid purification is done on an activated cellulose membrane. Helicase dependant amplification assays have been developed for detection of Mycoplasma genitalium and Trichomonas vaginalis. Two detection methods are being investigated, optical and magnetic resonance biosensors. The Arduino platform is used for automated fluidic and thermal control as well as communication of results.
Nucleic Acid Testing (NAT) promises rapid, sensitive and specific diagnosis of infectious, inheri... more Nucleic Acid Testing (NAT) promises rapid, sensitive and specific diagnosis of infectious, inherited and genetic disease. The next generation of diagnostic devices will interrogate the genetic determinants of such conditions at the point-of-care, affording clinicians prompt reliable diagnosis from which to guide more effective treatment. The complex biochemical nature of clinical samples, the low abundance of nucleic acid targets in the majority of clinical samples and existing biosensor technology indicate that some form of nucleic acid amplification will be required to obtain clinically relevant sensitivities from the small samples used in point-of-care testing (POCT). This publication provides an overview and thorough review of existing technologies for nucleic acid amplification. The different methods are compared and their suitability for POCT adaptation are discussed. Current commercial products employing isothermal amplification strategies are also investigated. In conclusion we identify the factors impeding the integration of the methods discussed in fully automated, sample-to-answer POCT devices.
Journal of Software Engineering and Applications, 2013
This paper presents a holistic methodology for the design of medical device software, which encom... more This paper presents a holistic methodology for the design of medical device software, which encompasses a new way of eliciting requirements, system design process, security design guideline, cloud architecture design, combinatorial testing process and agile project management. The paper uses point of care diagnostics as a case study where the software and hardware must be robust, reliable to provide accurate diagnosis of diseases. As software and software intensive systems are becoming increasingly complex, the impact of failures can lead to significant property damage, or damage to the environment. Within the medical diagnostic device software domain such failures can result in misdiagnosis leading to clinical complications and in some cases death. Software faults can arise due to the interaction among the software, the hardware, third party software and the operating environment. Unanticipated environmental changes and latent coding errors lead to operation faults despite the fact that usually a significant effort has been expended in the design, verification and validation of the software system. It is becoming increasingly more apparent that one needs to adopt different approaches, which will guarantee that a complex software system meets all safety, security, and reliability requirements, in addition to complying with standards such as IEC 62304. There are many initiatives taken to develop safety and security critical systems, at different development phases and in different contexts, ranging from infrastructure design to device design. Different approaches are implemented to design error free software for safety critical systems. By adopting the strategies and processes presented in this paper one can overcome the challenges in developing error free software for medical devices (or safety critical systems).
Advances in microfluidics and the introduction of isothermal nucleic acid amplification assays ha... more Advances in microfluidics and the introduction of isothermal nucleic acid amplification assays have resulted in a range of solutions for nucleic acid amplification tests suited for point of care and field use. However, miniaturisation of instrumentation for such assays has not seen such rapid advances and fluorescence based assays still depend on complex, bulky and expensive optics such as fluorescence microscopes, photomultiplier tubes and sensitive lens assemblies. In this work we demonstrate a robust, low cost platform for isothermal nucleic acid amplification on a microfluidic device. Using easily obtainable materials and commercial off-the-shelf components, we show real time fluorescence detection using a low cost photodiode and operational amplifier without need for lenses. Temperature regulation on the device is achieved using a heater fabricated with standard printed circuit board fabrication methods. These facile construction methods allow fabrications at a cost compatible with widespread deployment to resource poor settings.
consortium developing electronic self-testing instruments for sexually transmitted infections usi... more consortium developing electronic self-testing instruments for sexually transmitted infections using nucleic acid amplification testing (NAAT). A proprietary sample collection device has been designed to integrate directly with a microfluidic cartridge. Cell lysis was conducted using a chemical method and nucleic acid purification was done on an activated cellulose membrane. The microfluidic device incorporates passive mixing of the lysis-binding buffers and sample. Preliminary results have shown extraction efficiencies for this new membrane of 69 % and 57 % compared to the commercial Qiagen extraction method of 85 % and 59.4 % for 0.1ng/µL and 100ng/µL salmon sperm DNA spiked in phosphate buffered solution. Preliminary extraction experiments in the passive mixer cartridges with lysis and nucleic acid purification showed extraction efficiency around 80 % of the commercial Qiagen kit. Isothermal amplification was conducted using thermophillic helicase dependant amplification. A low co...
Advances in microfluidics and the introduction of isothermal nucleic acid amplification assays ha... more Advances in microfluidics and the introduction of isothermal nucleic acid amplification assays have resulted in a range of solutions for nucleic acid amplification tests suited for point of care and field use. However, miniaturisation of instrumentation for such assays has not seen such rapid advances and fluorescence based assays still depend on complex, bulky and expensive optics such as fluorescence microscopes, photomultiplier tubes and sensitive lens assemblies. In this work we demonstrate a robust, low cost platform for isothermal nucleic acid amplification on a microfluidic device. Using easily obtainable materials and commercial off-the-shelf components, we show real time fluorescence detection using a low cost photodiode and operational amplifier without need for lenses. Temperature regulation on the device is achieved using a heater fabricated with standard printed circuit board fabrication methods. These facile construction methods allow fabrications at a cost compatible with widespread deployment to resource poor settings.
Algae are considered to be a good source of alternative renewable energy for biofuel as up to 60%... more Algae are considered to be a good source of alternative renewable energy for biofuel as up to 60% of the world is covered in water and contains algae, chlorophyll and plankton, where each of these absorbs light and fixes carbon dioxide through photosynthesis [1]. Wavelength of the light source plays an important role in the algal growth. In this study, the experimental arrangement implemented to study the effects of using blue lasers as coherent light sources on the growth of Algae is explained in comparison to that of the white light LED irradiation. Three individual lasers having the following wavelengths (405 nm, 450 nm and 473 nm) were employed to generate photons with emission spectra matching the pigments' absorption spectra of Chlamydomonas reinhardtii algae; this was with a view to improving transportation of electrons within photosystems II and I, thus expecting an increase in cell division rate. C. reinhardtii algae effectively grew under exposure to both the blue lase...
A method has been developed using chitosan impregnated on membranes reducing the complexity of nu... more A method has been developed using chitosan impregnated on membranes reducing the complexity of nucleic acid extraction. This can be automated in a microfluidic system prior to nucleic acid amplification and detection. The procedure is pH dependent and involves the binding of DNA at pH 5.0 with DNA release at pH 9.0. The results showed extraction efficiencies of 63% and 82% for input samples of 100ng/µL and 0.1ng/µL respectively in comparison to benchtop Qiagen extraction results of 59% and 85%.
Antimicrobial resistance (AMR) is threatening modern medicine. While the primary cost of AMR is p... more Antimicrobial resistance (AMR) is threatening modern medicine. While the primary cost of AMR is paid in the healthcare domain, the agricultural and environmental domains are also reservoirs of resistant microorganisms and hence perpetual sources of AMR infections in humans. Consequently, the World Health Organisation and other international agencies are calling for surveillance of AMR in all three domains to guide intervention and risk reduction strategies. Technologies for detecting AMR that have been developed for healthcare settings are not immediately transferable to environmental and agricultural settings, and limited dialogue between the domains has hampered opportunities for cross-fertilisation to develop modified or new technologies. In this feature, we discuss the limitations of currently available AMR sensing technologies used in the clinic for sensing in other environments, and what is required to overcome these limitations.
A point of care device utilising Lab-on-a-Chip technologies that is applicable for biological pat... more A point of care device utilising Lab-on-a-Chip technologies that is applicable for biological pathogens was designed, fabricated and tested showing sample in to answer out capabilities. The purpose of the design was to develop a cartridge with the capability to perform nucleic acid extraction and purification from a sample using a chitosan membrane at an acidic pH. Waste was stored within the cartridge with the use of sodium polyacrylate to solidify or gelate the sample in a single chamber. Nucleic acid elution was conducted using the RPA amplification reagents (alkaline pH). Passive valves were used to regulate the fluid flow and a multiplexer was designed to distribute the fluid into six microchambers for amplification reactions. Cartridges were produced using soft lithography of silicone from 3D printed moulds, bonded to glass substrates. The isothermal technique, RPA is employed for amplification. This paper shows the results from two separate experiments: the first using the RPA control nucleic acid, the second showing successful amplification from Chlamydia Trachomatis. Endpoint analysis conducted for the RPA analysis was gel electrophoresis that showed 143 base pair DNA was amplified successfully for positive samples whilst negative samples did not show amplification. End point analysis for Chlamydia Trachomatis samples was fluorescence detection that showed successful detection of 1 copy/μL and 10 copies/μL spiked in a MES buffer.
Sustained observations of microbial dynamics are rare, especially in southern hemisphere waters. ... more Sustained observations of microbial dynamics are rare, especially in southern hemisphere waters. The Australian Marine Microbial Biodiversity Initiative (AMMBI) provides methodologically standardized, continental scale, temporal phylogenetic amplicon sequencing data describing Bacteria, Archaea and microbial Eukarya assemblages. Sequence data is linked to extensive physical, biological and chemical oceanographic contextual information. Samples are collected monthly to seasonally from multiple depths at seven sites: Darwin Harbour (Northern Territory), Yongala (Queensland), North Stradbroke Island (Queensland), Port Hacking (New South Wales), Maria Island (Tasmania), Kangaroo Island (South Australia), Rottnest Island (Western Australia). These sites span ~30° of latitude and ~38° longitude, range from tropical to cold temperate zones, and are influenced by both local and globally significant oceanographic and climatic features. All sequence datasets are provided in both raw and proce...
In the standard-care period, 40/41 (97.6%, 95% CI: 87.1-99.9) received treatment; 15 (37.5%) were... more In the standard-care period, 40/41 (97.6%, 95% CI: 87.1-99.9) received treatment; 15 (37.5%) were treated presumptively on the same day (due to symptoms/risk) and 25 (62.5%) were treated on the basis of the laboratory test result; and of these 44% were treated in 1-7 days, 44% in 8-14 days and 12% in 15+ days, with a median time-to-treatment of 8 days. Conclusion This site is already part of a strong and comprehensive STI control program run by NHC over 20 years. Early findings from TTANGO show further improvement in STI management were achieved with point-of-care tests, with treatment occurring on average 8 days sooner for those treated on the basis of a test result. Future analyses will include all 12 clinics and also assess if re-infections have reduced. Disclosure of interest statement No pharmaceutical grants were received in the development of this study. TTANGO is funded by a NHMRC project grant. The GeneXpert cartridges were purchased from Cepheid and Cepheid provided machines on loan for the duration of the study.
This paper presents the design of a modular point of care test platform that integrates a proprie... more This paper presents the design of a modular point of care test platform that integrates a proprietary sample collection device directly with a microfluidic cartridge. Cell lysis, within the cartridge, is conducted using a chemical method and nucleic acid purification is done on an activated cellulose membrane. The microfluidic device incorporates passive mixing of the lysis-binding buffers and sample using a serpentine channel. Results have shown extraction efficiencies for this new membrane of 69% and 57% compared to the commercial Qiagen extraction method of 85% and 59.4% for 0.1ng/µL and 100ng/µL salmon sperm DNA respectively spiked in phosphate buffered solution. Extraction experiments using the serpentine passive mixer cartridges incorporating lysis and nucleic acid purification showed extraction efficiency around 80% of the commercial Qiagen kit. Isothermal amplification was conducted using thermophillic helicase dependant amplification and recombinase polymerase amplification...
training could be improved with requests for a formalised attachment, formal qualification and gr... more training could be improved with requests for a formalised attachment, formal qualification and greater training in practical procedures. The BASHH GD SIG, in liaison with BASHH, aims to optimise GD training for registrars. Plans for improved resources are in progress, including a practical skills course and e-learning.
Brunel DoCLab is part of the esti2 consortium developing electronic self-testing instruments for ... more Brunel DoCLab is part of the esti2 consortium developing electronic self-testing instruments for sexually transmitted infections using nucleic acid amplification testing (NAAT). A proprietary sample collection device has been designed to integrate directly with a microfluidic cartridge. Cell lysis is conducted using a chemical method and nucleic acid purification is done on an activated cellulose membrane. Helicase dependant amplification assays have been developed for detection of Mycoplasma genitalium and Trichomonas vaginalis. Two detection methods are being investigated, optical and magnetic resonance biosensors. The Arduino platform is used for automated fluidic and thermal control as well as communication of results.
Nucleic Acid Testing (NAT) promises rapid, sensitive and specific diagnosis of infectious, inheri... more Nucleic Acid Testing (NAT) promises rapid, sensitive and specific diagnosis of infectious, inherited and genetic disease. The next generation of diagnostic devices will interrogate the genetic determinants of such conditions at the point-of-care, affording clinicians prompt reliable diagnosis from which to guide more effective treatment. The complex biochemical nature of clinical samples, the low abundance of nucleic acid targets in the majority of clinical samples and existing biosensor technology indicate that some form of nucleic acid amplification will be required to obtain clinically relevant sensitivities from the small samples used in point-of-care testing (POCT). This publication provides an overview and thorough review of existing technologies for nucleic acid amplification. The different methods are compared and their suitability for POCT adaptation are discussed. Current commercial products employing isothermal amplification strategies are also investigated. In conclusion we identify the factors impeding the integration of the methods discussed in fully automated, sample-to-answer POCT devices.
Journal of Software Engineering and Applications, 2013
This paper presents a holistic methodology for the design of medical device software, which encom... more This paper presents a holistic methodology for the design of medical device software, which encompasses a new way of eliciting requirements, system design process, security design guideline, cloud architecture design, combinatorial testing process and agile project management. The paper uses point of care diagnostics as a case study where the software and hardware must be robust, reliable to provide accurate diagnosis of diseases. As software and software intensive systems are becoming increasingly complex, the impact of failures can lead to significant property damage, or damage to the environment. Within the medical diagnostic device software domain such failures can result in misdiagnosis leading to clinical complications and in some cases death. Software faults can arise due to the interaction among the software, the hardware, third party software and the operating environment. Unanticipated environmental changes and latent coding errors lead to operation faults despite the fact that usually a significant effort has been expended in the design, verification and validation of the software system. It is becoming increasingly more apparent that one needs to adopt different approaches, which will guarantee that a complex software system meets all safety, security, and reliability requirements, in addition to complying with standards such as IEC 62304. There are many initiatives taken to develop safety and security critical systems, at different development phases and in different contexts, ranging from infrastructure design to device design. Different approaches are implemented to design error free software for safety critical systems. By adopting the strategies and processes presented in this paper one can overcome the challenges in developing error free software for medical devices (or safety critical systems).
Advances in microfluidics and the introduction of isothermal nucleic acid amplification assays ha... more Advances in microfluidics and the introduction of isothermal nucleic acid amplification assays have resulted in a range of solutions for nucleic acid amplification tests suited for point of care and field use. However, miniaturisation of instrumentation for such assays has not seen such rapid advances and fluorescence based assays still depend on complex, bulky and expensive optics such as fluorescence microscopes, photomultiplier tubes and sensitive lens assemblies. In this work we demonstrate a robust, low cost platform for isothermal nucleic acid amplification on a microfluidic device. Using easily obtainable materials and commercial off-the-shelf components, we show real time fluorescence detection using a low cost photodiode and operational amplifier without need for lenses. Temperature regulation on the device is achieved using a heater fabricated with standard printed circuit board fabrication methods. These facile construction methods allow fabrications at a cost compatible with widespread deployment to resource poor settings.
Uploads
Papers by Pascal Craw