02772nas a2200265 4500008004100000022001400041245009700055210006900152260000900221300001200230490000600242520198800248100002102236700002402257700002102281700002102302700001802323700001602341700001802357700001702375700001702392700002002409700001802429856005902447 2014 eng d a1932-620300aElectricity-free amplification and detection for molecular point-of-care diagnosis of HIV-1.0 aElectricityfree amplification and detection for molecular pointo c2014 ae1136930 v93 a
In resource-limited settings, the lack of decentralized molecular diagnostic testing and sparse access to centralized medical facilities can present a critical barrier to timely diagnosis, treatment, and subsequent control and elimination of infectious diseases. Isothermal nucleic acid amplification methods, including reverse transcription loop-mediated isothermal amplification (RT-LAMP), are well-suited for decentralized point-of-care molecular testing in minimal infrastructure laboratories since they significantly reduce the complexity of equipment and power requirements. Despite reduced complexity, however, there is still a need for a constant heat source to enable isothermal nucleic acid amplification. This requirement poses significant challenges for laboratories in developing countries where electricity is often unreliable or unavailable. To address this need, we previously developed a low-cost, electricity-free heater using an exothermic reaction thermally coupled with a phase change material. This heater achieved acceptable performance, but exhibited considerable variability. Furthermore, as an enabling technology, the heater was an incomplete diagnostic solution. Here we describe a more precise, affordable, and robust heater design with thermal standard deviation <0.5°C at operating temperature, a cost of approximately US$.06 per test for heater reaction materials, and an ambient temperature operating range from 16°C to 30°C. We also pair the heater with nucleic acid lateral flow (NALF)-detection for a visual readout. To further illustrate the utility of the electricity-free heater and NALF-detection platform, we demonstrate sensitive and repeatable detection of HIV-1 with a ß-actin positive internal amplification control from processed sample to result in less than 80 minutes. Together, these elements are building blocks for an electricity-free platform capable of isothermal amplification and detection of a variety of pathogens.
1 aSingleton, Jered1 aOsborn, Jennifer, L1 aLillis, Lorraine1 aHawkins, Kenneth1 aGuelig, Dylan1 aPrice, Will1 aJohns, Rachel1 aEbels, Kelly1 aBoyle, David1 aWeigl, Bernhard1 aLaBarre, Paul uhttp://www.microfluidicsciences.com/drupal/?q=node/20002192nas a2200205 4500008004100000022001400041245010900055210006900164260001500233300001100248490000900259520154600268100002101814700001901835700001601854700001601870700001801886700002301904856005901927 2013 ENG d a0277-786X00aInstrument-free exothermic heating with phase change temperature control for paper microfluidic devices.0 aInstrumentfree exothermic heating with phase change temperature c2013 Mar 9 a86150R0 v86153 aMany infectious diseases, as well as some cancers, that affect global health are most accurately diagnosed through nucleic acid amplification and detection. There is a great need to simplify nucleic acid-based assay systems for use in global health in low-resource settings as well as in settings that do not have convenient access to laboratory staff and equipment such as doctors' offices and home care settings. In developing countries, unreliable electric power, inadequate supply chains, and lack of maintenance for complex diagnostic instruments are all common infrastructure shortfalls. Many elements of instrument-free, disposable, nucleic acid amplification assays have been demonstrated in recent years. However, the problem of instrument-free, low-cost, temperature-controlled chemical heating remains unsolved. In this paper we present the current status and results of work towards developing disposable, low-cost, temperature-controlled heaters designed to support isothermal nucleic acid amplification assays that are integrated with a two-dimensional paper network. Our approach utilizes the heat generated through exothermic chemical reactions and controls the heat through use of engineered phase change materials to enable sustained temperatures required for nucleic acid amplification. By selecting appropriate exothermic and phase change materials, temperatures can be controlled over a wide range, suitable for various isothermal amplification methods, and maintained for over an hour at an accuracy of +/- 1°C.
1 aSingleton, Jered1 aZentner, Chris1 aBuser, Josh1 aYager, Paul1 aLaBarre, Paul1 aWeigl, Bernhard, H uhttp://www.microfluidicsciences.com/drupal/?q=node/20502000nas a2200205 4500008004100000022001400041245014400055210006900199260001300268300001400281490000700295520132200302100001601624700001801640700002301658700001301681700001801694700002301712856005901735 2013 ENG d a1001-653800aMolecular diagnostics in a teacup: Non-Instrumented Nucleic Acid Amplification (NINA) for rapid, low cost detection of Salmonella enterica.0 aMolecular diagnostics in a teacup NonInstrumented Nucleic Acid A c2013 Apr a1162-11680 v583 aWe report on the use of a novel non-instrumented platform to enable a Loop Mediated isothermal Amplification (LAMP) based assay for Salmonella enterica. Heat energy is provided by addition of a small amount (<150 g) of boiling water, and the reaction temperature is regulated by storing latent energy at the melting temperature of a lipid-based engineered phase change material. Endpoint classification of the reaction is achieved without opening the reaction tube by observing the fluorescence of sequence-specific FRET-based assimilating probes with a simple handheld fluorometer. At or above 22°C ambient temperature the non-instrumented devices could maintain reactions above a threshold temperature of 61°C for over 90 min-significantly longer than the 60 min reaction time. Using the simple format, detection limits were less than 20 genome copies for reactions run at ambient temperatures ranging from 8 to 36°C. When used with a pre-enrichment step and non-instrumented DNA extraction device, trace contaminations of Salmonella in milk close to 1 CFU/mL could be reliably detected. These findings illustrate that the non- instrumented amplification approach is a simple, viable, low-cost alternative for field-based food and agricultural diagnostics or clinical applications in developing countries.
1 aKubota, Ryo1 aLaBarre, Paul1 aWeigl, Bernhard, H1 aLi, Yong1 aHaydock, Paul1 aJenkins, Daniel, M uhttp://www.microfluidicsciences.com/drupal/?q=node/20402744nas a2200373 4500008004100000022001400041245009900055210006900154260000900223300001100232490000600243520168200249653002301931653001601954653001501970653002101985653001202006653001902018653001002037653001102047653004202058653002602100653001502126653001602141100002102157700002202178700001702200700002102217700001702238700002002255700001802275700001802293856005902311 2012 eng d a1932-620300aIsothermal amplification using a chemical heating device for point-of-care detection of HIV-1.0 aIsothermal amplification using a chemical heating device for poi c2012 ae314320 v73 aBACKGROUND: To date, the use of traditional nucleic acid amplification tests (NAAT) for detection of HIV-1 DNA or RNA has been restricted to laboratory settings due to time, equipment, and technical expertise requirements. The availability of a rapid NAAT with applicability for resource-limited or point-of-care (POC) settings would fill a great need in HIV diagnostics, allowing for timely diagnosis or confirmation of infection status, as well as facilitating the diagnosis of acute infection, screening and evaluation of infants born to HIV-infected mothers. Isothermal amplification methods, such as reverse-transcription, loop-mediated isothermal amplification (RT-LAMP), exhibit characteristics that are ideal for POC settings, since they are typically quicker, easier to perform, and allow for integration into low-tech, portable heating devices.
METHODOLOGY/SIGNIFICANT FINDINGS: In this study, we evaluated the HIV-1 RT-LAMP assay using portable, non-instrumented nucleic acid amplification (NINA) heating devices that generate heat from the exothermic reaction of calcium oxide and water. The NINA heating devices exhibited stable temperatures throughout the amplification reaction and consistent amplification results between three separate devices and a thermalcycler. The performance of the NINA heaters was validated using whole blood specimens from HIV-1 infected patients.
CONCLUSION: The RT-LAMP isothermal amplification method used in conjunction with a chemical heating device provides a portable, rapid and robust NAAT platform that has the potential to facilitate HIV-1 testing in resource-limited settings and POC.
10aAIDS Serodiagnosis10aDNA Primers10aDNA, Viral10aEquipment Design10aHeating10aHIV Infections10aHIV-110aHumans10aNucleic Acid Amplification Techniques10aPoint-of-Care Systems10aRNA, Viral10aTemperature1 aCurtis, Kelly, A1 aRudolph, Donna, L1 aNejad, Irene1 aSingleton, Jered1 aBeddoe, Andy1 aWeigl, Bernhard1 aLaBarre, Paul1 aOwen, Michele uhttp://www.microfluidicsciences.com/drupal/?q=node/20701785nas a2200169 4500008004100000022001400041245008200055210006900137260001600206490000900222520124900231100001801480700001701498700002101515700002001536856005901556 2011 ENG d a0277-786X00aInstrument-free nucleic acid amplification assays for global health settings.0 aInstrumentfree nucleic acid amplification assays for global heal c2011 May 160 v80293 aMany infectious diseases that affect global health are most accurately diagnosed through nucleic acid amplification and detection. However, existing nucleic acid amplification tests are too expensive and complex for most low-resource settings. The small numbers of centralized laboratories that exist in developing countries tend to be in urban areas and primarily cater to the affluent. In contrast, rural area health care facilities commonly have only basic equipment and health workers have limited training and little ability to maintain equipment and handle reagents.(1) Reliable electric power is a common infrastructure shortfall. In this paper, we discuss a practical approach to the design and development of non-instrumented molecular diagnostic tests that exploit the benefits of isothermal amplification strategies. We identify modular instrument-free technologies for sample collection, sample preparation, amplification, heating, and detection. By appropriately selecting and integrating these instrument-free modules, we envision development of an easy to use, infrastructure independent diagnostic test that will enable increased use of highly accurate molecular diagnostics at the point of care in low-resource settings.
1 aLaBarre, Paul1 aBoyle, David1 aHawkins, Kenneth1 aWeigl, Bernhard uhttp://www.microfluidicsciences.com/drupal/?q=node/21001962nas a2200217 4500008004100000022001400041245011800055210006900173260000900242300001000251490000600261520127900267100001601546700001801562700002101580700001701601700002301618700002101641700002301662856005901685 2011 ENG d a2162-643X00aNon-Instrumented Nucleic Acid Amplification (NINA) for Rapid Detection of Ralstonia solanacearum Race 3 Biovar 2.0 aNonInstrumented Nucleic Acid Amplification NINA for Rapid Detect c2011 a69-800 v43 aWe report on the use of a non-instrumented device for the implementation of a loop-mediated amplification (LAMP) based assay for the select-agent bacterial-wilt pathogen Ralstonia solanacearum race 3 biovar 2. Heat energy is generated within the device by the exothermic hydration of calcium oxide, and the reaction temperature is regulated by storing latent energy at the melting temperature of a renewable lipid-based engineered phase-change material. Endpoint detection of the LAMP reaction is achieved without opening the reaction tube by observing the fluorescence of an innovative FRET-based hybridization probe with a simple custom fluorometer. Non-instrumented devices could maintain reactions near the design temperature of 63°C for at least an hour. Using this approach DNA extracted from the pathogen could be detected at fewer than ten copies within a 25 μL reaction mix, illustrating the potential of these technologies for simple, powerful agricultural diagnostics in the field. Furthermore, the assay was just as reliable when implemented in a tropical environment at 31°C as it was when implemented in an air-conditioned lab maintained at 22°C, illustrating the potential value of the technology for field conditions in the tropics and subtropics.
1 aKubota, Ryo1 aLaBarre, Paul1 aSingleton, Jered1 aBeddoe, Andy1 aWeigl, Bernhard, H1 aAlvarez, Anne, M1 aJenkins, Daniel, M uhttp://www.microfluidicsciences.com/drupal/?q=node/21403019nas a2200313 4500008004100000022001400041245015500055210006900210260000900279300001100288490000600299520199700305653002502302653001602327653002002343653003602363653004202399653002602441653002402467100001802491700002402509700001702533700001902550700001902569700002102588700001702609700002002626856005902646 2011 eng d a1932-620300aA simple, inexpensive device for nucleic acid amplification without electricity-toward instrument-free molecular diagnostics in low-resource settings.0 asimple inexpensive device for nucleic acid amplification without c2011 ae197380 v63 aBACKGROUND: Molecular assays targeted to nucleic acid (NA) markers are becoming increasingly important to medical diagnostics. However, these are typically confined to wealthy, developed countries; or, to the national reference laboratories of developing-world countries. There are many infectious diseases that are endemic in low-resource settings (LRS) where the lack of simple, instrument-free, NA diagnostic tests is a critical barrier to timely treatment. One of the primary barriers to the practicality and availability of NA assays in LRS has been the complexity and power requirements of polymerase chain reaction (PCR) instrumentation (another is sample preparation).
METHODOLOGY/PRINCIPAL FINDINGS: In this article, we investigate the hypothesis that an electricity-free heater based on exothermic chemical reactions and engineered phase change materials can successfully incubate isothermal NA amplification assays. We assess the heater's equivalence to commercially available PCR instruments through the characterization of the temperature profiles produced, and a minimal method comparison. Versions of the prototype for several different isothermal techniques are presented.
CONCLUSIONS/SIGNIFICANCE: We demonstrate that an electricity-free heater based on exothermic chemical reactions and engineered phase change materials can successfully incubate isothermal NA amplification assays, and that the results of those assays are not significantly different from ones incubated in parallel in commercially available PCR instruments. These results clearly suggest the potential of the non-instrumented nucleic acid amplification (NINA) heater for molecular diagnostics in LRS. When combined with other innovations in development that eliminate power requirements for sample preparation, cold reagent storage, and readout, the NINA heater will comprise part of a kit that should enable electricity-free NA testing for many important analytes.
10aDeveloping Countries10aElectricity10aHot Temperature10aMolecular Diagnostic Techniques10aNucleic Acid Amplification Techniques10aPlasmodium falciparum10aReference Standards1 aLaBarre, Paul1 aHawkins, Kenneth, R1 aGerlach, Jay1 aWilmoth, Jared1 aBeddoe, Andrew1 aSingleton, Jered1 aBoyle, David1 aWeigl, Bernhard uhttp://www.microfluidicsciences.com/drupal/?q=node/20902408nas a2200277 4500008004100000022001400041245012900055210006900184260000900253300001100262490000900273520153100282653001901813653001201832653001101844653002401855653003601879653004201915100001801957700001701975700001901992700001902011700002102030700002002051856005902071 2010 eng d a1557-170X00aNon-instrumented nucleic acid amplification (NINA): instrument-free molecular malaria diagnostics for low-resource settings.0 aNoninstrumented nucleic acid amplification NINA instrumentfree m c2010 a1097-90 v20103 aWe have achieved the first complete, non-instrumented nucleic acid amplification test (NAAT) using a calcium oxide heat source thermally linked to an engineered phase change material. These two components alone maintain a thermal profile suitable for the loop-mediated isothermal amplification assay. Starting with computational fluid dynamics analysis, we identified nominal geometry for the exothermic reaction chamber, phase change material chamber, thermal insulation, and packaging. Using this model, we designed and fabricated an alpha prototype assay platform. We have verified the function of this multi-pathogen-capable platform with both fluorescent and visual turbidity indications using samples spiked with malaria DNA. Both the exothermically heated platform samples and samples heated on a Perkin-Elmer GeneAmp9600 thermocycler were first incubated at 62°C for 45 minutes, then heated to 95°C to terminate enzyme activity, then analyzed. Results from the exothermically heated, non-instrumented platform were comparable to those from the thermocycler. These developments will enable point-of-care diagnostics using accurate NAATs which until now have required a well-equipped laboratory. The aim of this research is to provide pathogen detection with NAAT-level sensitivity in low-resource settings where assays such as immunochromatographic strip tests are successfully used but where there is no access to the infrastructure and logistics required to operate and maintain instrument-based diagnostics.
10aDNA, Protozoan10aHeating10aHumans10aMalaria, Falciparum10aMolecular Diagnostic Techniques10aNucleic Acid Amplification Techniques1 aLaBarre, Paul1 aGerlach, Jay1 aWilmoth, Jared1 aBeddoe, Andrew1 aSingleton, Jered1 aWeigl, Bernhard uhttp://www.microfluidicsciences.com/drupal/?q=node/21501793nas a2200217 4500008004100000022001400041245008500055210006900140260001300209300001400222490000600236520111400242653004101356653002501397653001801422100002001440700002101460700001801481700001701499856005901516 2008 eng d a1473-019700aTowards non- and minimally instrumented, microfluidics-based diagnostic devices.0 aTowards non and minimally instrumented microfluidicsbased diagno c2008 Dec a1999-20140 v83 aIn many health care settings, it is uneconomical, impractical, or unaffordable to maintain and access a fully equipped diagnostics laboratory. Examples include home health care, developing-country health care, and emergency situations in which first responders are dealing with pandemics or biowarfare agent release. In those settings, fully disposable diagnostic devices that require no instrument support, reagent, or significant training are well suited. Although the only such technology to have found widespread adoption so far is the immunochromatographic rapid assay strip test, microfluidics holds promise to expand the range of assay technologies that can be performed in formats similar to that of a strip test. In this paper, we review progress toward development of disposable, low-cost, easy-to-use microfluidics-based diagnostics that require no instrument at all. We also present examples of microfluidic functional elements--including mixers, separators, and detectors--as well as complete microfluidic devices that function entirely without any moving parts and external power sources.
10aDiagnostic Techniques and Procedures10aDisposable Equipment10aMicrofluidics1 aWeigl, Bernhard1 aDomingo, Gonzalo1 aLaBarre, Paul1 aGerlach, Jay uhttp://www.microfluidicsciences.com/drupal/?q=node/218