An Investigation of the Applicability of Sugar and Wax Valves for rapid molecular diagnostics

Paper-based Loop-mediated Isothermal Amplification (LAMP) of nucleic acids is a low-cost molecular diagnostic method to detect biological pathogens and has been successfully combined with lateral flow devices. This invention is a low-cost visual detection technique of various disease. However, existing assays require multiple user-activated steps and are relatively complicated to use at the point of care due to steps like the heating process. This project aimed to investigate an ideal valve for integrating LAMP onto the lateral flow assay. An ideal valve needs to be able to stop the fluid flow for 20-60 minutes under 65 degree to complete the amplification and release the water flow automatically. Sugar and wax were examined as potential valve materials. The sugar valve was tested by pipetting various concentrations of sucrose solution onto paper strips and drying the strips. For these valves, the time it took for water to dissolve the sugar was the total valve timing. The wax valve was made by dipping the paper strips into melted wax and then cooling. The melting point of the wax was higher than 65 degree so that after the heating process, the wax valve could be “opened” by increasing the temperature and allowing the fluid to flow. The sugar valves lacked repeatability due to environmental conditions such as humidity and difficulty of distributing the sugar solution evenly on the paper strips. In contrast, the wax valve was demonstrated to have incredible reliability and control over fluid flow. We managed to stop the water flow completely with wax valve and open it within 2 minutes. This paper proved that wax has potential to be a good valve for paper-based loop-mediated isothermal amplification

Quantification of Analyte Concentration from a Paper-Based Lateral Flow Assay Device Using Reflective Sensors

Paper-based point-of-care (POC) diagnostics is a growing field in global health due to the extreme portability, accuracy, affordability, and ease of use of these tests. Advancements in recent years have led to more accurate detection and improved functionality using multistep molecular diagnostics. Many such assays utilize lateral flow detection strips for visualization of diagnostic results by eye, which limits the results to qualitative Yes/No readouts. This project focused on combining recent developments in paper-based POC diagnostics to develop and optimize an in-house built quantitative paper-based diagnostic reader for lateral flow detection in low-resource settings. Initially different sensors, including photocell sensors, reflective IR sensors, and light-to-frequency converters were tested to optimize detection method in terms of accuracy, precision, and affordability. After the detection method was determined, the sensor was calibrated using hCG (pregnancy) test strips from Wondfo® Co. to compare the in-house detection method to a standard image quantification through the ImageJ application. These analyses will be used to calibrate the circuit to relate the light intensity calculated to the concentration of analyte (hCG) present in the sample as well as determine the lower limit of detection (LLOD). The IR reflective sensor and the light-to-frequency converter performed best due to the wide detection output range, adaptability for dark environments, and consistency in detection. We will develop and calibrate both methods for future use. The detection methods determined in this project provide a platform for a multistep diagnostic device for DNA for uses in environmental, food, and health safety, and can be applied to other paper-based diagnostics for accurate quantified results

Stimuli Responsive Fluidics Controls on a Paper-Based Bacterial Detection Platform

Infectious diseases are the leading causes of death around the world. Point-of-care devices using nucleic acid amplification are sensitive enough to diagnose these diseases, however, often require complex and time-intensive sample preparation steps that are not integrated with the detection process. A rapid, sensitive, and integrated sample-to-result diagnostic device will permit disease treatment planning at the point-of-care. Paper-based detection assays are a promising platform to integrate the sample preparation and detection, with minimal infrastructure, equipment, and user involvement. To integrate sample preparation with detection on paper-based assays, timing and delivery of sample fluid flow needs to be controlled. Here we use thermally responsive materials (e.g. wax) to create a micro valve on a nitrocellulose membrane in order to automate fluid flow and minimize user involvement. The ease-of-fabrication, lot-to-lot variability and consistency of the dispensing methods are compared. After multiple trials, dispensing the wax material, PureTemp 68X, using a stamp made of polydimethylsiloxane (PDMS) is able to direct the sample flow with the highest consistency. Thermally responsive valves fabricated by stamping PureTemp 68X are found to block the sample fluid to flow for a sustained time when cool. When heated above the melting temperature (68°C), the valve opens and allows fluid flow without interfering with downstream assay binding reactions. These valves can be actuated multiple times simply by heating and cooling again. As a proof of concept, we use the valve to control sample delivery time of nucleic acid amplicons in a lateral flow immunoassay. Amplicons dispensed onto one side of the assay are incubated and bind to gold nanoparticles. They are then released through the valve by applying heat to open the valve. Nanoparticle nucleic acid amplicons will bind to recognition antibodies at the far side of the valve and become visible bands for detection

Lysis and Amplifciation of Neonatal Sepsis Causing Pathogens

Neonatal sepsis, resulting from a bloodstream infection within the first few weeks of life, is the leading cause of newborn deaths worldwide. The gold standard of neonatal sepsis diagnosis requires a blood culture to identify the infecting bacteria, however require days of incubation, expensive equipment, and expertise. Any delay in diagnosis is critical, as the condition can be treated easily if appropriate antibiotics are administered promptly. A low-cost, rapid, and sensitive diagnostic test would enable more timely treatment and lead to better patient outcomes with fewer required resources. Point-of-care, nucleic acid amplification assays are a promising alternative to blood culture that quickly deliver sensitive results with minimal sample volume. However, these require isolated and purified template DNA from the pathogenic bacteria, a task that is difficult to achieve in a field setting. This study sought to develop a simple one-step lysis and amplification protocol for three common bacterial causes of neonatal sepsis, Streptococcus agalacitae, Klebsiella pnemoniae, and Staphylococcus aureus. The combined efficacy of enzymes, proteinase K and achromopeptidase (ACP), and heat to lyse each of these bacteria for direct DNA amplification was examined. Results showed that all three strains could be effectively lysed by applying 1 U of ACP and incubating for 10 minutes at 37° C. No adverse effects were seen in amplification reactions containing ACP if the compound was inactivated prior to amplification at 85° C for 2 minutes. The demonstrated effectiveness of ACP in rapid bacterial lysis validates its usefulness in a point-of-care device for neonatal sepsis

Fully Dried Two-Dimensional Paper Network for Enzymatically Enhanced Detection of Nucleic Acid Amplicons

Two-dimensional paper networks (2DPNs) have enabled the use of paper-based platforms to perform multistep immunoassays for detection of pathogenic diseases at the point-of-care. To date, however, detection has required the user to provide multiple signal enhancement solutions and been limited to protein targets. We solve these challenges by using mathematical equations to guide the device design of a novel 2DPN, which leverages multiple fluidic inputs to apply fully dried solutions of hydrogen peroxide, diaminobenzidine, and horseradish peroxidase signal enhancement reagents to enhance the limit-ofdetection of numerous nucleic acid products. Upon rehydration in our unique 2DPN design, the dried signal enhancement solution reduces the limit-of-detection (LOD) of the device to 5 × 1011 nucleic acid copies/mL without increasing false positive detection. Our easy-to-use device retains activity after 28 days of dry storage and produces reliable signal enhancement 40 min after sample application. The fully integrated device demonstrated versatility in its ability to detect double-stranded and single-stranded DNA samples, as well as peptide nucleic acids

Fluidic Control with Wax Valves for Paper-based Diagnostics

Paper-fluidic devices are a common platform for point-of-care disease detection in under-resourced areas because of their low cost and minimal instrumentation requirements. Limited fluidic control in paper-fluidic devices has hindered the incorporation of multistep reactions that are necessary for more sensitive disease detection. One potential fluidic control mechanism is the incorporation of thermally actuated wax valves to separate assay stages. Such valving would expand the detection capabilities of these devices by permitting fluid obstruction for sustained reactions and facilitating controlled volume release within a fully-automated, self-contained device. Despite the potential to exploit wax valves for innovative paper-fluidic diagnostics, a thorough, quantitative analysis of how they can best be used has not been performed. Here, in parallel macroscopic and microscopic analyses, we show that wax valves’ geometry and surface area in paper test strips influence flow behavior when thermally actuated. Macroscopic analysis evaluated the flow rate past the valves of the visible fluid front across the width of the membrane; microscopic analysis used particle image velocimetry to evaluate trends in particle flow before and after valve actuation. Preliminary results indicate that geometry and size influence valve opening times and the rate of fluid flow past the valves. Future analyses will compare the macroscopic and microscopic velocity profiles in various assay spaces and times to provide quantitative insight to the inner workings of paper-fluidic devices. This information will facilitate intelligent and efficient design of multistep paper-fluidic detection technologies with potential applications in lateral flow immunoassays, two-dimensional paper networks, and other point-of-care diagnostics

Thin Film Cocaine Sensors

Over 7 million Americans suffer from a drug use disorder and up to 60% of individuals treated for addiction will ultimately relapse. We are developing ultra-thin film electrodes on a wearable substrate for a sensor that can detect minute amounts of cocaine in sweat droplets secreted from the skin. This will enable wearable drug monitoring for personalized rehabilitation treatment plans and improve long-term addiction recovery rates. The current research focuses on developing a thin-film sensor that can be applied directly to the skin. First a layer of PVP (poly4-vinylphenol) was prepared and then spun coated onto a piece of glass. This adhesive coated glass acted as a substrate for assembling the sensors. Silver reference electrodes were generated by evaporating silver through a stencil, which contained 1mm by 10mm slits, in a vacuum sealed chamber. The resulting electrodes were tested to verify their conductivity, stability, and reactivity. Upon successfully demonstrating these characteristics, a second stencil was made to evaporate both gold and silver to make a working electrode surface that will react with a cocaine solution, making the first prototype that can successfully detect cocaine

A paperfluidic platform to detect Neisseria gonorrhoeae in clinical samples

Globally, the microbe Neisseria gonorrhoeae (NG) causes 106 million newly documented sexually transmitted infections each year. Once appropriately diagnosed, NG infections can be readily treated with antibiotics, but high-risk patients often do not return to the clinic for treatment if results are not provided at the point of care. A rapid, sensitive molecular diagnostic would help increase NG treatment and reduce the prevalence of this sexually transmitted disease. Here, we report on the design and development of a rapid, highly sensitive, paperfluidic device for point-of-care diagnosis of NG. The device integrates patient swab sample lysis, nucleic acid extraction, thermophilic helicase-dependent amplification (tHDA), an internal amplification control (NGIC), and visual lateral flow detection within an 80 min run time. Limits of NG detection for the NG/NGIC multiplex tHDA assay were determined within the device, and clinical performance was validated retroactively against qPCR-quantified patient samples in a proof-of-concept study. This paperfluidic diagnostic has a clinically relevant limit of detection of 500 NG cells per device with analytical sensitivity down to 10 NG cells per device. In triplicate testing of 40 total urethral and vaginal swab samples, the device had 95% overall sensitivity and 100% specificity, approaching current laboratory-based molecular NG diagnostics. This diagnostic platform could increase access to accurate NG diagnoses to those most in need.This work was funded by the National Institute of Health National Institute of Allergy and Infectious Diseases award number R01 AI113927 to Boston University and the NIH National Institute of Biomedical and Bioengineering award number U54 EB007958 to Johns Hopkins University. (R01 AI113927 - National Institute of Health National Institute of Allergy and Infectious Diseases; U54 EB007958 - NIH National Institute of Biomedical and Bioengineering)Accepted manuscrip

Smartphone-Based Microscope for Pathogen Detection

Vibrio cholerae is a water and food borne bacteria that causes cholera, a severe acute diarrheal disease, when ingested and when left untreated, can cause patient death within hours. Currently there is a lack of both sensitive and rapid portable detection technologies of V. cholerae for testing water and food samples. Combining nucleic acid amplification and particle diffusometry present an alternative detection method for V. cholerae in under 30 minutes, but the process requires an expensive laboratory microscope. In this work, we develop a smartphone-based microscope to detect V. cholerae DNA in environmental water samples using particle diffusometry. A modular iPhone case is designed with a detachable cartridge and an integrated ball lens to image a microfluidic sample slide. This sample slide is essential for performing the DNA amplification assay and contains the microparticles necessary for imaging and particle diffusometry measurements. The ball lenses to image these particles have diameters of 1.0, 0.79, and 0.5 millimeters and are tested in conjunction with the iPhone 6 camera to determine optimal magnification and focal length. We demonstrate the ability to detect the Brownian motion of 1.0 micrometer particles using a 0.5 millimeter ball lens with brightfield imaging. This field-portable imaging platform provides the capability to perform rapid detection of V. cholerae in the field, and in the future, can be applied toward detecting other diseases at the point of care

Design and development of an integrated mHealth platform to improve kangaroo mother care in Kenya

Background and Significance: There are 15 million preterm births a year. Premature babies suffer the highest rates of newborn mortality, occurring primarily in low/middle-income countries (LMICs). Neonatal hypothermia (low body temperature) is a life-threatening complication, which is prevented by Kangaroo Mother Care (KMC), but in Kenya, a profound shortage of health workers and lack of resources are barriers to KMC. Our international team has developed an integrated platform (educational and data collection apps + biomedical device) to improve the implementation of KMC in health facilities. Methods: From August 2020 – February 2021, a multi-disciplinary team from the United States and Kenya utilized agile development (weekly scrum meetings) and human-and user-centered design techniques to develop high-fidelity wireframes (Figma) of Android apps which are designed to integrate with a patented self-warming biomedical device (US10390630B2; NG/PT/IC/2016/053394) that utilizes wireless sensors to track KMC babies, continuously monitor infant vital signs, and display physiological data on mobile phones/tablets. Results: High-fidelity wireframes have been developed for two user interfaces of an integrated app, NeoRoo. The NeoRoo-Family app is for KMC parents; the NeoRoo-HealthWorker app is built for nurses and doctors. NeoRoo-Family provides parental caregivers with: (a) automated monitoring of key vital signs for their baby; (c) ability to alert a clinician as needed; (c) tracking of KMC metrics and goals, such as number of hours of skin-toskin care completed in a week; and (d) educational resources for evidence-based newborn care. The NeoRoo- HealthWorker app interface enables clinicians to: (a) simultaneously track breathing, heart rate, temperature, and oxygen saturation for multiple KMC infants in real-time; (b) review each infant’s past clinical history and vital signs trends; (c) receive automated and parent-generated alerts; (d) support harmonized dissemination of key educational messages to families. Conclusions: By providing education, continuous thermal support, and integrated, automated vital signs monitoring for premature babies, via the NeoRoo mHealth platform, we hope to better equip parents and health workers in Kenya to: (1) prevent hypothermia; (2) automatically monitor vital signs in newborns; (3) track key KMC metrics; (4) promote more effective task-sharing among KMC teams. On-going work includes participatory design interviews and a usability assessment