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

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

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

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

Towards the use of a smartphone imaging-based tool for point-of-care detection of asymptomatic low-density malaria parasitaemia

Background: Globally, there are over 200 million cases of malaria annually and over 400,000 deaths. Early and accurate detection of low-density parasitaemia and asymptomatic individuals is key to achieving the World Health Organization (WHO) 2030 sustainable development goals of reducing malaria-related deaths by 90% and eradication in 35 countries. Current rapid diagnostic tests are neither sensitive nor specific enough to detect the low parasite concentrations in the blood of asymptomatic individuals. Methods: Here, an imaging-based sensing technique, particle diffusometry (PD), is combined with loop mediated isothermal amplification (LAMP) on a smartphone-enabled device to detect low levels of parasitaemia often associated with asymptomatic malaria. After amplification, PD quantifies the Brownian motion of fluorescent nanoparticles in the solution during a 30 s video taken on the phone. The resulting diffusion coefficient is used to detect the presence of Plasmodium DNA amplicons. The coefficients of known negative samples are compared to positive samples using a one-way ANOVA post-hoc Dunnett's test for confirmation of amplification. Results: As few as 3 parasite/µL of blood was detectable in 45 min without DNA extraction. Plasmodium falciparum parasites were detected from asymptomatic individuals' whole blood samples with 89% sensitivity and 100% specificity when compared to quantitative polymerase chain reaction (qPCR). Conclusions: PD-LAMP is of value for the detection of low density parasitaemia especially in areas where trained personnel may be scarce. The demonstration of this smartphone biosensor paired with the sensitivity of LAMP provides a proof of concept to achieve widespread asymptomatic malaria testing at the point of care

Enabling the Development and Deployment of Next Generation Point-of-Care Diagnostics

<p>Enabling the Development and Deployment of Next Generation Point-of-Care Diagnostics</p

Quantitation of Trastuzumab and an Antibody to SARS-CoV-2 in Minutes Using Affinity Membranes in 96-Well Plates

This article is made available for unrestricted research re-use and secondary analysis in any form or be any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.Quantitation of therapeutic monoclonal antibodies (mAbs) in human serum could ensure that patients have adequate levels of mAbs for effective treatment. This research describes the use of affinity, glass-fiber membranes in a 96-well-plate format for rapid (<5 min) quantitation of the therapeutic mAb trastuzumab and a mAb against the SARS-CoV-2 spike protein. Adsorption of a poly(acrylic acid)-containing film in membrane pores and activation of the -COOH groups in the film enable covalent-linking of affinity peptides or proteins to the membrane. Passage of mAb-containing serum through the affinity membrane results in mAb capture within 1 min. Subsequent rinsing, binding of a secondary antibody conjugated to a fluorophore, and a second rinse yield mAb-concentration-dependent fluorescence intensities in the wells. Calibration curves established from analyses on different days have low variability and allow determination of mAb levels in separately prepared samples with an average error <10%, although errors in single-replicate measurements may reach 40%. The assays can occur in diluted serum with physiologically relevant mAb concentrations, as well as in undiluted serum. Thus, the combination of 96-well plates containing affinity membranes, a microplate reader, and a simple vacuum manifold affords convenient mAb quantitation in <5 min

KickStat: A Coin-Sized Potentiostat for High-Resolution Electrochemical Analysis

The demand for wearable and point-of-care devices has led to an increase in electrochemical sensor development to measure an ever-increasing array of biological molecules. In order to move from the benchtop to truly portable devices, the development of new biosensors requires miniaturized instrumentation capable of making highly sensitive amperometric measurements. To meet this demand, we have developed KickStat, a miniaturized potentiostat that combines the small size of the integrated Texas Instruments LMP91000 potentiostat chip (Texas Instruments, Dallas, TX, USA) with the processing power of the ARM Cortex-M0+ SAMD21 microcontroller (Microchip Technology, Chandler, AZ, USA) on a custom-designed 21.6 mm by 20.3 mm circuit board. By incorporating onboard signal processing via the SAMD21, we achieve 1 mV voltage increment resolution and an instrumental limit of detection of 4.5 nA in a coin-sized form factor. This elegant engineering solution allows for high-resolution electrochemical analysis without requiring extensive circuitry. We measured the faradaic current of an anti-cocaine aptamer using cyclic voltammetry and square wave voltammetry and demonstrated that KickStat&rsquo;s response was within 0.6% of a high-end benchtop potentiostat. To further support others in electrochemical biosensors development, we have made KickStat&rsquo;s design and firmware available in an online GitHub repository