Design and assembly of microfluidic paper-based analytical devices (μPADS) for the quantification of nitrite and nitrate in saliva

Nos últimos anos, investigadores, com o auxílio de novas tecnologias, têm trabalhado para o desenvolvimento de técnicas e dispositivos de diagnóstico e tratamento mais práticos e económicos. Neste trabalho foram desenvolvidos dois dispositivos microfluídicos analíticos baseados em papel (μPADs) para a determinação dos aniões nitrito e nitrato em amostras de saliva humana, para auxílio no diagnóstico de doenças associadas à sua presença. Para desenvolver estes μPADs foram realizados vários estudos de desenho e construção, incluindo um teste de interferências e estudos de estabilidade. A estrutura final do μPAD para a determinação de anião nitrito consistiu em duas camadas de discos de papel de filtro com 9.5 mm de diâmetro numa bolsa de plastificação, em que a última camada continha 5 μL de reagente de Griess. Este μPAD permitiu a determinação de nitrito num intervalo de 5 – 220 μM, cujos limites de deteção e quantificação foram 0.05 μM e 0.17 μM, respetivamente. A estrutura final do μPAD para a determinação de anião nitrato consistiu em três camadas de papel de filtro numa bolsa de plastificação, em que a primeira camada continha zinco em pó e a última camada 10 μL de reagente de Griess. Este μPAD permitiu a determinação de anião nitrato num intervalo de 0.2 – 1.2 mM, cujos limites de deteção e quantificação foram 0.08 mM e 0.27 mM, respetivamente. Ambos os μPADs se mostraram estáveis quando armazenados em vácuo (durante pelo menos 7 dias no caso do μPAD de nitrito, e durante no máximo 3 dias no caso do μPAD de nitrato) e, após colocação da amostra, os μPADs de nitrito e nitrato poderiam ser digitalizados até 4 e 2 horas depois, respetivamente. Por último, para validar este método, os resultados obtidos com o μPAD de nitrito foram comparados com os correspondentes aos obtidos pelo método colorimétrico de referência, e não foram encontradas diferenças estatisticamente significativas entre os dois métodos. Logo, foi possível concluir que os μPADs desenvolvidos demonstraram possuir propriedades promissoras para a determinação de NOX em amostras de saliva, principalmente porque são dispositivos sensíveis, portáteis, simples e económicos, que custam menos de 50 cêntimos cada.In the last few years, researchers, with the help of new and advanced technologies, have been working towards the development of more practical and more affordable, diagnostic and treatment devices and techniques. In this work, two different Microfluidic Paper-based Analytical Devices (μPADs) were developed for the determination of nitrite and nitrate in human saliva samples to aid in the diagnosis of some diseases and health conditions associated with these ions. To develop these nitrite and nitrate μPADs, several studies were performed to optimize the design and construction, including an interference assessment and stability studies. The final structure of developed μPAD for the nitrite determination consisted of two layers of 9.5 mm diameter filter paper disks within a plastic laminating pouch, in which the bottom layer contained 5 μL of Griess reagent. This μPAD allowed a nitrite determination in a range of 5 - 220 μM with limits of detection and quantification of 0.05 μM and 0.17 μM, respectively. The nitrate μPAD final structure consisted of three layers of filter paper, also within a plastic laminating pouch, in which the top layer contained the zinc powder and the bottom layer contained 10 μL of Griess reagent. This μPAD allowed a nitrate determination in the range 0.2 – 1.2 mM with limits of detection and quantification of 0.08 mM and 0.27 mM, respectively. Both of the μPADs were stable when stored in vacuum (the nitrite μPAD for at least 7 days and the nitrate μPAD for a maximum of 3 days) and, after the sample placement, the nitrite and nitrate μPADs could be scanned within the first 4 and 2 hours, respectively. Finally, to validate this method, nitrite μPAD measurements were compared with the ones obtained from the standard colorimetric method and there were no statistically significant differences between these two methods. So, it was possible to conclude that the developed μPADs exhibited promising properties for NOX determinations in saliva samples, especially because they are sensitive, portable, simple and affordable devices that cost less than 50 cents each

Salivary calcium determination with a specially developed microfluidic paper-based device for point-of-care analysis

The calcium monitoring in the body not only anticipates several potential diseases (osteoporosis, kidney stones or high blood pressure) but also helps to improve target therapies and follow-up the patient's health status. Calcium monitoring is essential for the diagnosis of one of the most common endocrine disorders worldwide, namely hyperparathyroidism. So, in this work, a new Point-of-care test (POC-test) using a microfluidic paper-based analytical device (μPAD) for calcium quantification in saliva samples is described. The developed μPAD was based on the colorimetric reaction between calcium and cresolphthalein complexone (CPC) which forms an intense purple colour product. The developed device enabled calcium quantification in the range of 0.27–4.50 mmol/L (11.0–180 mg/L) with a detection limit of 80 µmol/L (3.2 mg/L). The accuracy of the developed μPAD was confirmed by analysing saliva samples (#10) and comparing the results obtained with the atomic absorption spectrometry reference procedure; the relative deviation between the two sets of results was below 10 %. A correlation between salivary calcium content and calcium content in blood was established and it was possible to conclude that salivary calcium concentrations above 1.55 mmol/L is an indicator of hypercalcemia. The developed device was stable for 2 weeks when stored at room temperature in vacuum conditions.info:eu-repo/semantics/publishedVersio

New microfluidic paper-based analytical device for iron determination in urine samples

Iron is an important micronutrient involved in several mechanisms in the human body and can be an important biomarker. In this work, a simple and disposable microfluidic paper-based analytical device (µPAD) was developed for the quantification of iron in urine samples. The detection was based on the colorimetric reaction between iron(II) and bathophenanthroline and the reduction of iron(III) to iron(II) with hydroxylamine. The developed µPAD enabled iron determination in the range 0.07–1.2 mg/L, with a limit of detection of 20 µg/L and a limit of quantification of 65 µg/L, thus suitable for the expected values in human urine. Additionally, targeting urine samples, the potential interference of the samples color was overcome by incorporating a sample blank assessment for absorbance subtraction. Stability studies revealed that the device was stable for 15 days prior to usage and that the formed colored product was stable for scanning up to 3 h. The accuracy of the developed device was established by analyzing urine samples (#26) with the developed µPAD and with the atomic absorption spectrometry method; the relative deviation between the two sets of results was below 9.5%. Graphical abstract: [Figure not available: see fulltext.]info:eu-repo/semantics/acceptedVersio

Three-Dimensional Paper-Based Microfluidic Device for Assays of Protein and Glucose in Urine

The first step in curing a disease is being able to detect the disease effectively. Paper-based microfluidic devices are biodegradable and can make diagnosing diseases cost-effective and easy in almost all environments. We created a three-dimesnional (3D) paper device using wax printing fabrication technique and basic principles of origami. This design allows for a versatile fabrication technique over previously reported patterning of SU-8 photoresist on chromatography paper by employing a readily available wax printer. The design also utilizes multiple colorimetric assays that can accommodate one or more analytes including urine, blood, and saliva. In this case to demonstrate the functionality of the 3D paper-based microfluidic system, a urinalysis of protein and glucose assays is conducted. The amounts of glucose and protein introduced to the device are found to be proportional to the color change of each assay. This color change was quantified by use of Adobe Photoshop. Urine samples from participants with no pre-existing health conditions and one person with diabetes were collected and compared against synthetic urine samples with predetermined glucose and protein levels. Utilizing this method, we were able to confirm that both protein and glucose levels were in fact within healthy ranges for healthy participants. For the participant with diabetes, glucose was found to be above the healthy range while the protein level was in the healthy range

Anal Chem

Microfluidic paper-based analytical devices (\u3bcPADs) are simple but powerful analytical tools that are gaining significant recent attention due to their many advantages over more traditional monitoring tools. These include being inexpensive, portable, pump-free, and having the ability to store reagents. One major limitation of these devices is slow flow rates, which are controlled by capillary action in the hydrophilic pores of cellulosic paper. Recent investigations have advanced the flow rates in \u3bcPADs through the generation of a gap or channel between two closely spaced paper sheets. This multilayered format has opened up \u3bcPADs to new applications and detection schemes, where large gap sizes (>300 \u3bcm) provide at least 169 7 faster flow rates than single-layer \u3bcPADs, but do not conform to established mathematical models for fluid transport in porous materials, such as the classic Lucas-Washburn equation. In the present study, experimental investigations and analytical modeling are applied to elucidate the driving forces behind the rapid flow rates in these devices. We investigate a range of hypotheses for the systems fluid dynamics and establish a theoretical model to predict the flow rate in multilayered \u3bcPADs that takes into account viscous dissipation within the paper. Device orientation, sample addition method, and the gap height are found to be critical concerns when modeling the imbibition in multilayered devices.R01 OH010662/OH/NIOSH CDC HHS/United StatesR01OH010662/ACL/ACL HHS/United StatesR33 ES024719/ES/NIEHS NIH HHS/United States2020-11-10T00:00:00Z31276368PMC7653499866

Detection of C-Reactive Protein Using an ELISA Immunodot as a Proof-of-Concept for Paper Microfluidics

Medicine relies heavily on diagnostic testing. Before the end of 2019 – the beginning of 2020, the modernized world took for granted accurate and available diagnostic tests. The COVID-19 pandemic taught the world, even the wealthiest countries, how fragile human health can become when tests are lacking. The assumption of available testing and the confidence in test results has been seriously challenged. With these challenges, Point-of-Care (PoC) tests has transgressed medicine and science to include politics, finance, and humanity at its core. This Bard senior project is rooted in the science of a proof-of-concept paper-based ELISA Immunodot assay for the detection of C-reactive protein (CRP). CRP can be identified at varying blood concentrations found in humans physiology and disease. CRP testing is used for clinical diagnoses millions of times per month in the United States. The results confirm that the ELISA Immunodot can both distinguish CRP+ and CRP- standards and semi-quantitively predict the CRP concentration of the standard. The ability to relate the intensity of the CRP colorimetric output to a standard CRP concentration has potential applicability in future medical testing

Novel Integration of Conductive-ink Circuitry with a Paper-based Microfluidic Battery as an All-printed Sensing Platform

The addition of powered components for active assays into paper-based analytical devices opens new opportunities for medical and environmental analysis in resource-limited applications. Current battery designs within such devices have yet to adopt a ubiquitous circuitry material, necessitating investigation into printed circuitry for scalable platforms. In this study, a microfluidic battery was mated with silver-nanoparticle conductive ink to prototype an all-printed sensing platform. A multi-layer, two-cell device was fabricated, generating 200 μA of direct electrical current at 2.5 V sustained for 16 minutes with a power loss of less than 0.1% through the printed circuitry. Printed circuitry traces exhibited resistivity of 75 to 211 10-5 Ω m. Resistance of the printed traces increased upwards of 200% depending on fold angle and directionality. X-ray diffraction confirmed the presence of face-centered cubic silver after sintering printed traces for 30 minutes at 150°C in air. A conductivity threshold was mapped and an ink concentration of 0.636 μL mm-3 was identified as the lower limit for optimal electrical performance

Lab Chip

Microfluidic paper-based analytical devices (\u3bcPADs) are a versatile and inexpensive point-of-care (POC) technology, but their widespread adoption has been limited by slow flow rates and the inability to carry out complex in field analytical measurements. In the present work, we investigate multilayer \u3bcPADs as a means to generate enhanced flow rates within self-pumping paper devices. Through optical and electrochemical measurements, the fluid dynamics are investigated and compared to established flow theories within \u3bcPADs. We demonstrate a 3c145-fold increase in flow rate (velocity = 1.56 cm s|, volumetric flow rate = 1.65 mL min|, over 5.5 cm) through precise control of the channel height in a 2 layer paper device, as compared to archetypical 1 layer \u3bcPAD designs. These design considerations are then applied to a self-pumping sequential injection device format, known as a three-dimensional paper network (3DPN). These 3DPN devices are characterized through flow injection analysis of a ferrocene complex and anodic stripping detection of cadmium, exhibiting a 5 7 enhancement in signal compared to stationary measurements.R01 OH010662/OH/NIOSH CDC HHS/United StatesR01OH010662/ACL/ACL HHS/United States2020-03-14T00:00:00Z29431751PMC70715577359vault:3510