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Background: Optimization of automated insulin delivery (AID) settings is required to achieve desirable glycemic outcomes. We evaluated safety and efficacy of a computerized system to initialize and adjust insulin delivery settings for the t:slim X2 insulin pump with Control-IQ technology in adults with type 1 diabetes (T1D)., Methods: After a 2-week continuous glucose monitoring (CGM) run-in period, adults with T1D using multiple daily injections (MDI) (N = 33, mean age 36.1 years, 57.6% female, diabetes duration 19.7 years) were transitioned to 13 weeks of Control-IQ technology usage. A computerized algorithm generated recommendations for initial pump settings (basal rate, insulin-to-carbohydrate ratio, and correction factor) and weekly follow-up settings to optimize glycemic outcomes. Physicians could override the automated settings changes for safety concerns., Results: Time in range 70 to 180 mg/dL improved from 45.7% during run-in to 69.1% during the last 30 days of Control-IQ use, a median improvement of 18.8% (95% confidence interval [CI]: 13.6-23.9, P < .001). This improvement was evident early in the study and was sustained over 13 weeks. Time <70 mg/dL showed a gradual decreasing trend over time. Percentage of participants achieving HbA1c <7% went from zero at baseline to 55% at study end ( P < .001). Only six of the 318 automated settings adaptations (1.9%) were overridden by study investigators., Conclusions: Computerized initiation and adaptation of Control-IQ technology settings from baseline MDI therapy was safe in adults with T1D. The use of this simplified system for onboarding and optimizing Control-IQ technology may be useful to increase uptake of AID and reduce staff and patient burden in clinical care., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: VNS has received research grants through University of Colorado from Tandem Diabetes Care, Insulet, NovoNordisk, Alexion, JDRF and NIH. He has received fees through University of Colorado for speaking or consulting work for Dexcom, Insulet, Tandem Diabetes Care, Embecta, NovoNordisk and Medscape. HKA has received research grants through University of Colorado from Tandem Diabetes Care, Medtronic, Dexcom, Eli Lilly, Mannkind. He has received consulting fees through University of Colorado from Tandem Diabetes Care, Medtronic and Dexcom. AT, NP, AW, ES, KC, LM, KW, LF, RSK, LM, SH, AC, and JEP are employees of Tandem Diabetes Care, Inc.

The use of lateral flow assays to detect nucleic acid targets has many applications including point-of-care diagnostics, environmental monitoring, and food safety. A sandwich format, similar to that in protein immunoassays, is often used to capture the target nucleic acid sequence with an immobilized complementary strand anchored to a substrate, and then to visualize this event using a complementary label nucleic acid bound to a nanoparticle label. A critical component of high-sensitivity nucleic acid detection is to utilize high-density capture surfaces for the effective capture of target nucleic acid. Multiple methods have been reported, including the use of streptavidin-based protein anchors that can be adsorbed to the lateral flow substrate and that can utilize the high-affinity streptavidin-biotin linkage to bind biotinylated nucleic acid capture sequences for subsequent target nucleic acid binding. However, these protein anchors have not been systematically characterized for use in the context of nucleic acid detection. In this work, we characterize several protein-based anchors on nitrocellulose for (i) capturing the robustness of the attachment of the protein anchor, (ii) capturing nucleic acid density, and (iii) targeting nucleic acid capture. Further, we demonstrate the signal gains in target nucleic acid hybridization made by increasing the density of capture nucleic acid on a nitrocellulose substrate using multiple applications of protein loading onto nitrocellulose. Finally, we use our high-density capture surfaces to demonstrate high-sensitivity nucleic acid detection in a lateral flow assay (in the context of a SARS-CoV-2 sequence), achieving a LOD of approximately 0.2 nM.

Carbamazepine (CBZ), a drug prescribed to prevent seizures in people with epilepsy, has a narrow therapeutic range such that patients would greatly benefit from personalized drug dosage recommendations. Saliva is an excellent sample for personalized monitoring of CBZ levels because saliva CBZ concentration correlates with the free concentration of CBZ in blood, and can be collected non-invasively. CBZ level quantification using electrochemical detection has been demonstrated in a variety of electrode systems and samples, however, human saliva presents a particular challenge in terms of its complex composition that can result in signal interference via a high background current at the potentials of interest for CBZ detection. Previous demonstrations of electrochemical detection of CBZ in saliva have included rigorous pre-treatment of the sample using centrifugation and high levels of dilution, which is not compatible with lower-resource field settings for patient monitoring of CBZ levels. In this work, we systematically investigate several strategies to improve the detection of CBZ in a background of undiluted human saliva using polymeric laminate-based devices with stencil-printed carbon electrodes; (i) adding the anionic surfactant sodium dodecyl sulfate to the saliva, (ii) filtering saliva to remove larger molecular weight species, (iii) plasma pretreatment of the device electrodes, and (iv) incubation of the sample on the electrodes. These methods enabled the quantification of therapeutically-relevant concentrations of CBZ in a background of human saliva without the need for saliva preprocessing like dilution.

Paper microfluidics is a rapidly growing subfield of microfluidics in which paper-like porous materials are used to create analytical devices that are well-suited for use in field applications. 3D printing technology has the potential to positively affect paper microfluidic device development by enabling tools and methods for the creation of devices with well-defined and tunable fluidic networks of porous matrices for high performance signal generation. This critical review focuses on the progress that has been made in using 3D printing technologies to advance the development of paper microfluidic devices. We describe printing work in three general categories: (i) solid support structures for paper microfluidic device components; (ii) channel barrier definition in existing porous materials; and (iii) porous channels for capillary flow, and discuss their value in advancing paper microfluidic device development. Finally, we discuss major areas of focus for highest impact on the next generation of paper microfluidics devices.

Background: Direct Acting Antivirals (DAAs) represent a large improvement in the treatment of chronic hepatitis C, resulting in <90% sustained virological response (SVR). There are no reports on the real-world DAA response for Mexico and few reports exist for Latin America. The aim of the study was to report SVR, and immediate benefits with the DAA treatments sofosbuvir, ledispavir, with/without ribavirin (SOF/LDV ± RBV) and ombitasvir, paritaprevir, ritonavir, dasabuvir with/without RBV (OBV/PTV/r/DSV ± RBV) in patients with viral genotype 1a or 1b, and who did not respond to previous peginterferon/ribavirin (PegIFNα2a+RBV) therapy., Methods: A descriptive, ambispective, longitudinal study was conducted. A cohort of 261 adult patients received PegIFNα2a+RBV therapy before 2014; 167 (64%) did not respond, 83 of these were subsequently treated with SOF/LDV ± RBV or OBV/PTV/r/DSV ± RBV. Child-Pugh-Score (CPS), Fibrosis-4 (FIB-4), and AST to Platelet Ratio Index (APRI) were evaluated before and after treatment., Results: SVR with PegIFNα2a+RBV was 36%, and 97.5% with DAAs. CPS, FIB-4 and APRI improved significantly after DAA treatment, mainly because of liver transaminase reduction., Conclusions: DAA treatment showed excellent SVR rates in Mexican patients who had not responded to PegIFNα2a+RBV therapy. Improvement in CPS, FIB-4 and APRI without improvement in fibrosis was observed in cirrhotic and non-cirrhotic patients, as well as considerable reduction in liver transaminases, which suggests a reduction in hepatic necroinflammation., Competing Interests: The authors declare there are no competing interests., (©2021 Melendez-Mena et al.)

Background: High-level antibiotic consumption plays a critical role in the selection and spread of extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E) in the ICU. Implementation of a stewardship program including a restrictive antibiotic policy was evaluated with respect to ESBL-E acquisition (carriage and infection)., Methods: We implemented a 2-year, before-and-after intervention study including all consecutive adult patients admitted for > 48 h in the medical-surgical 26-bed ICU of Guadeloupe University Hospital (French West Indies). A conventional strategy period (CSP) including a broad-spectrum antibiotic as initial empirical treatment, followed by de-escalation (period before), was compared to a restrictive strategy period (RSP) limiting broad-spectrum antibiotics and shortening their duration. Antibiotic therapy was delayed and initiated only after microbiological identification, except for septic shock, severe acute respiratory distress syndrome and meningitis (period after). A multivariate Cox proportional hazard regression model adjusted on propensity score values was performed. The main outcome was the median time of being ESBL-E-free in the ICU. Secondary outcome included all-cause ICU mortality., Results: The study included 1541 patients: 738 in the CSP and 803 in the RSP. During the RSP, less patients were treated with antibiotics (46.8% vs. 57.9%; p < 0.01), treatment duration was shorter (5 vs. 6 days; p < 0.01), and administration of antibiotics targeting anaerobic pathogens significantly decreased (65.3% vs. 33.5%; p < 0.01) compared to the CSP. The incidence of ICU-acquired ESBL-E was lower (12.1% vs. 19%; p < 0.01) during the RSP. The median time of being ESBL-E-free was 22 days (95% CI 16-NA) in the RSP and 18 days (95% CI 16-21) in the CSP. After propensity score weighting and adjusted analysis, the median time of being ESBL-E-free was independently associated with the RSP (hazard ratio, 0.746 [95% CI 0.575-0.968]; p = 0.02, and hazard ratio 0.751 [95% CI 0.578-0.977]; p = 0.03, respectively). All-cause ICU mortality was lower in the RSP than in the CSP (22.5% vs. 28.6%; p < 0.01)., Conclusions: Implementation of a program including a restrictive antibiotic strategy is feasible and is associated with less ESBL-E acquisition in the ICU without any worsening of patient outcome., (© 2021. The Author(s).)

The degradation of biochemical reagents on the timescale of weeks can severely limit the utility of microfluidic assays intended for field use, and is a challenging aspect of microfluidic device development in general. Our study focuses on the evaluation of the dry storage stability of three types of reagents: (i) the colorimetric reagents nitroblue tetrazolium and 1-methoxy-5-methylphenazinium methylsulfate, (ii) the enzyme phenylalanine dehydrogenase, and (iii) the coenzyme β-nicotinamide adenine dinucleotide hydrate, within the context of a phenylalanine monitoring device. We have demonstrated stable dry storage of each of the reagents, over the time span of approximately one month. Drying the colorimetric reagents under nitrogen was found to largely suppress reagent degradation and the appearance of nonspecific signal, while the enzyme and coenzyme retained activity when stored dry for a month without additional processing or chemical additives. Finally, phenylalanine monitoring devices with all three reagent types dried down and stored for 15 days showed comparable functionality to devices containing freshly-dried reagents - a key milestone to enable future clinical testing.

Background: Patients on maintenance hemodialysis are particularly vulnerable to infection and hospitalization from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Due to immunocompromised patients and the clustering that occurs in outpatient dialysis units, the seroprevalence of COVID-19 antibodies in this population is unknown and has significant implications for public health. Also, little is known about their risk factors for hospitalization., Methods: Three outpatient maintenance hemodialysis units affiliated with a major teaching hospital in the New York area were studied. We determined rates of SARS-CoV-2 positivity via nasopharyngeal, real-time, reverse-transcriptase PCR (RT-PCR); SARS-CoV-2 IgG seropositivity; hospitalization; and mortality., Results: Of 367 patients, 28% had either SARS-CoV-2 seropositivity or PCR positivity. Prevalence across the three respective units was 7%, 32%, and 70%. Those who were either antibody or PCR positive were significantly younger (65 versus 69 years, P =0.05), and had a higher prevalence of Black race (43% versus 30%, P =0.001) and Hispanic ethnicity (32% versus 12%, P <0.001) compared with those who tested negative. Higher positivity rates were also observed among those who took taxis and ambulettes to and from dialysis, compared with those who used personal transportation. Antibodies were detected in all of the patients with a positive PCR result who underwent serologic testing. Of those that were seropositive, 32% were asymptomatic. The hospitalization rate on the basis of either antibody or PCR positivity was 35%, with a hospital mortality rate of 33%. Aside from COPD, no other variables were more prevalent in patients who were hospitalized., Conclusions: We observed significant differences in rates of COVID-19 infection within three outpatient dialysis units, with universal seroconversion. Among patients with ESKD, rates of asymptomatic infection appear to be high, as do hospitalization and mortality rates., Competing Interests: N. Ali reports receiving honoraria from CareDx. J. Divers reports receiving honoraria from the National Institutes of Health and Oak Ridge Associated Universities. P. Dutka reports receiving research funding from Akebia, Bayer, GlaxoSmithKline, and Novo Nordisk; being on the editorial board of the Nephrology Nursing Journal; and having consultancy agreements with, receiving honoraria from, and being on a speakers bureau for Rockwell Medical. C. Grant reports receiving research funding from Novo Nordisk. N. Masani reports having ownership interest in AbbVie, Bristol Meyers Squibb, General Electric, and Siemens; receiving research funding from Cara Therapeutics, GlaxoSmithKline, and Novo Nordisk; being a scientific advisor for, or member of, Rockwell Medical; and being a member of Renal Physicians Association. All remaining authors have nothing to disclose., (Copyright © 2021 by the American Society of Nephrology.)

C-reactive protein levels may have clinical value in monitoring different phases of healing and in identifying possible states of infection. As a critical step to further investigate this potential connection, we have demonstrated a lateral flow assay (LFA) for canine CRP level assessment in wound exudate that could be used as a tool in the veterinary clinic setting. In the rational design of our cCRP LFA, we have characterized LFA performance for sequential delivery mode vs. the more common premixed delivery mode using several metrics including dynamic range, sensitivity, limit of detection, and time to result. Although the sequential mode assay results indicated modestly improved signal (3-14%) and limit of detection (in the low ng/mL range for both) for this set of cCRP immunoassay reagents, the premixed mode assay's shorter run time with one less delivery step was chosen for use in this application in which analyte levels are substantially elevated. We have defined a straightforward wound exudate processing procedure that includes centrifugation to extract exudate from canine patient bandages, and subsequent sample dilution for cCRP quantification by our LFA. And, we have demonstrated that our cCRP LFA provides comparable cCRP concentrations to that of gold-standard ELISA performed on the same clinical wound exudate, and serum/plasma samples. Finally, we have highlighted some next steps in the assessment of cCRP as a biomarker for wound healing and infection., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Metal toxicity is a common problem in crop species worldwide. Some metals are naturally toxic, whereas others such as manganese (Mn) are essential micro-nutrients for plant growth but can become toxic when in excess. Changes in the composition of the xylem sap, which is the main pathway for ion transport within the plant, is therefore vital to understanding the plant's response(s) to metal toxicity. In this study we have assessed the effects of exposure of tomato roots to excess Mn on the protein profile of the xylem sap, using a shotgun proteomics approach. Plants were grown in nutrient solution using 4.6 and 300 µM MnCl 2 as control and excess Mn treatments, respectively. This approach yielded 668 proteins reliably identified and quantified. Excess Mn caused statistically significant (at p ≤ 0.05) and biologically relevant changes in relative abundance (≥2-fold increases or ≥50% decreases) in 322 proteins, with 82% of them predicted to be secretory using three different prediction tools, with more decreasing than increasing (181 and 82, respectively), suggesting that this metal stress causes an overall deactivation of metabolic pathways. Processes most affected by excess Mn were in the oxido-reductase, polysaccharide and protein metabolism classes. Excess Mn induced changes in hydrolases and peroxidases involved in cell wall degradation and lignin formation, respectively, consistent with the existence of alterations in the cell wall. Protein turnover was also affected, as indicated by the decrease in proteolytic enzymes and protein synthesis-related proteins. Excess Mn modified the redox environment of the xylem sap, with changes in the abundance of oxido-reductase and defense protein classes indicating a stress scenario. Finally, results indicate that excess Mn decreased the amounts of proteins associated with several signaling pathways, including fasciclin-like arabinogalactan-proteins and lipids, as well as proteases, which may be involved in the release of signaling peptides and protein maturation. The comparison of the proteins changing in abundance in xylem sap and roots indicate the existence of tissue-specific and systemic responses to excess Mn. Data are available via ProteomeXchange with identifier PXD021973.

There is a need for a common, shared definition of rehabilitation to conduct systematic reviews and identify relevant systematic reviews for knowledge translation purposes, which is an important task of Cochrane Rehabilitation. The present paper aimed to introduce and compare existing health-related definitions of rehabilitation and to propose core aspects that should characterize a new and workable definition of rehabilitation that is able to serve both as the basis for internal communication and identity work and for external communication. We have conducted a PubMed literature search on current definitions that have been published since the launch of WHO's ICF in 2001. Definitions were analyzed by framing questions to which the definitions provide answers. Nine definitions were included in the analysis. Rehabilitation has been defined as a process, as a set or bundle of interventions, and as a health strategy. The main beneficiaries were mainly related to the presence of disability, however, no specific means or interventions in rehabilitation could be identified. The definitions provided varying answers to the questions "by whom…," "where…," and "when…" and additionally identified certain conditions for rehabilitation. The present analysis can serve as a valuable source of information for developing a Cochrane Rehabilitation definition of rehabilitation.

Competing Interests: Die Autorinnen/Autoren geben an, dass kein Interessenkonflikt besteht.

A promising application of paper microfluidics is the translation of gold-standard multi-step laboratory tests to a disposable paper-based format for decentralized diagnostic or therapeutic testing. This often entails conversion of bench-top processing of macro-volume samples to the processing of micro-volume samples within a porous matrix, and requires detailed characterization of fluid and reagent interactions within the porous material(s) of the device. The current study focuses on rational device design through the characterization of fluid and reagent interactions in polysulfone and glass fiber substrates for multi-step sample processing. Specifically, we demonstrate how the characterization of fluidic compatibility between substrates, chemical compatibility between reagents and substrates, sample pH, and sample transport can be used to inform device design in the context of a two-reaction detection scheme for phenylalanine in porous materials. Finally, we demonstrate detection of phenylalanine from human whole blood, and discuss the multiple strengths of the current design over a previous version.

In this study, the response of 6-month-old cocoa (Theobroma cacao L.) seedlings to elevated CO 2 concentration [ECO 2 ], elevated temperature [ET], and their interaction with water deficit stress was studied in an open top chamber (OTC). Each OTC was maintained at chamber control (400 ppm CO 2 ), [ECO 2 ] 550 ppm, [ECO 2 ] 700 ppm, ET 3 °C above chamber control, and ET 3 °C + [ECO 2 ] 550 ppm. Inside each OTC, a set of plants received moisture at 100% FC, while the other set was at 50% FC, which was the water deficit stress treatment. Increasing the CO 2 concentration in cocoa increased photosynthesis (Pn) by 27%, which resulted in high biomass accumulation, thus improving the whole plant water use efficiency (WUE). The impact of high temperature (T max ), around 39 °C in ET treatment against 36 °C in chamber control, is quite severe on Pn, leaf Ψ, and biomass accumulation. Similarly, water deficit at 50% FC resulted in the leaf Ψ reducing to - 14.06 bars at which Pn, leaf area, and biomass were significantly reduced. [ECO 2 ] could ameliorate the negative effect of high temperature and water deficit stress to certain extent. However, the relative response of cocoa seedlings to [ECO 2 ] in improving Pn, leaf Ψ, biomass, and WUE was greater under 50% FC compared to plants at 100% FC suggested additional advantage of [ECO 2 ] to cocoa under water limited conditions.

Although fabrics have great promise as substrates for use in wearable precision health applications, there has been relatively little attention focused on the development of control tools suitable for use in fabrics. Fluid control tools in fabric would enable the automation of multi-step sample processing on the device, reducing the need for off-chip sample handling. In this study, we describe the operation and characterization of a wax-based valving method with an integrated resistive heater in fabric for automated fluid delivery. The combination of wax-transfer-printed wax barrier and stencil-printed conductive ink heating element in fabric is a novel approach for achieving fluid control. We demonstrate robust valve operation and a rapid valve response time, and quantify the reproducibility of fluid flow through replicate valves. Further, we characterize wax redistribution in fabric using optical methods and scanning electron microscope imaging. Finally, we demonstrate valve utility in the context of on-device incubation in a fabric-based device for electrochemical glucose sensing. With a fabrication method that is compatible with a variety of substrates, this valving method has broad applicability.

The aim of this work was to assess the effects of manganese (Mn) toxicity on the proteome of tomato roots using two proteomic approaches, shotgun and two-dimensional electrophoresis. The shotgun approach yielded 367 reliable proteins, whereas the 2-DE approach detected 340 consistent spots. The 2-DE method found 54 proteins changing in relative abundance in the excess Mn treatment, whereas the shotgun detected changes in 118 proteins. Only 7% of the differential proteins were found by both methods, illustrating their complementary nature. Metabolic pathways most affected were protein metabolism, oxido-reductases and signaling. Results support that Mn toxicity alters the protein turnover and impairs energy production in roots, leading to changes in glycolysis, pyruvate metabolism, TCA and oxidative phosphorylation. Excess Mn also induced changes in peroxidases and hydrolases participating in cell wall lignification and suberization and activated plant defense mechanisms, with changes occurring via pathogenesis-related proteins as well as peroxidases. Finally, Mn toxicity elicited regulatory mechanisms and affected the abundance of root nutrient reservoir proteins. The overall analysis of the differential root proteome upon Mn toxicity suggests a general slowdown of metabolic activities, especially energy production, cell wall integrity and protein turnover, which occurs in parallel with increases in stress related proteins., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This article contains consolidated proteomic data obtained from xylem sap collected from tomato plants grown in Fe- and Mn-sufficient control, as well as Fe-deficient and Mn-deficient conditions. Data presented here cover proteins identified and quantified by shotgun proteomics and Progenesis LC-MS analyses: proteins identified with at least two peptides and showing changes statistically significant (ANOVA; p ≤ 0.05) and above a biologically relevant selected threshold (fold ≥ 2) between treatments are listed. The comparison between Fe-deficient, Mn-deficient and control xylem sap samples using a multivariate statistical data analysis (Principal Component Analysis, PCA) is also included. Data included in this article are discussed in depth in the research article entitled "Effects of Fe and Mn deficiencies on the protein profiles of tomato ( Solanum lycopersicum) xylem sap as revealed by shotgun analyses" [1]. This dataset is made available to support the cited study as well to extend analyses at a later stage.

Goodpasture antigen-binding protein (GPBP) is an exportable 1 Ser/Thr kinase that induces collagen IV expansion and has been associated with chemoresistance following epithelial-to-mesenchymal transition (EMT). Here we demonstrate that cancer EMT phenotypes secrete GPBP (mesenchymal GPBP) which displays a predominant multimeric oligomerization and directs the formation of previously unrecognized mesh collagen IV networks (mesenchymal collagen IV). Yeast two-hybrid (YTH) system was used to identify a 260 SHCIE 264 motif critical for multimeric GPBP assembly which then facilitated design of a series of potential peptidomimetics. The compound 3-[4''-methoxy-3,2'-dimethyl-(1,1';4',1'')terphenyl-2''-yl]propionic acid, or T12, specifically targets mesenchymal GPBP and disturbs its multimerization without affecting kinase catalytic site. Importantly, T12 reduces growth and metastases of tumors populated by EMT phenotypes. Moreover, low-dose doxorubicin sensitizes epithelial cancer precursor cells to T12, thereby further reducing tumor load. Given that T12 targets the pathogenic mesenchymal GPBP, it does not bind significantly to normal tissues and therapeutic dosing was not associated with toxicity. T12 is a first-in-class drug candidate to treat cancer by selectively targeting the collagen IV of the tumor cell microenvironment., Competing Interests: CONFLICTS OF INTEREST J.S, F.R and F.R-R are co-founders and stockholders of Fibrostatin, S.L. that is expanding GPBP inhibitors including T12 and hN26 into regulatory development. J.S acts as Chief Scientific Officer of Fibrostatin, S.L. The other authors declare that they have no competing interest other than being Fibrostatin, S.L. employees when indicated.

The aim of this work was to study the effects of Fe and Mn deficiencies on the xylem sap proteome of tomato using a shotgun proteomic approach, with the final goal of elucidating plant response mechanisms to these stresses. This approach yielded 643 proteins reliably identified and quantified with 70% of them predicted as secretory. Iron and Mn deficiencies caused statistically significant and biologically relevant abundance changes in 119 and 118 xylem sap proteins, respectively. In both deficiencies, metabolic pathways most affected were protein metabolism, stress/oxidoreductases and cell wall modifications. First, results suggest that Fe deficiency elicited more stress responses than Mn deficiency, based on the changes in oxidative and proteolytic enzymes. Second, both nutrient deficiencies affect the secondary cell wall metabolism, with changes in Fe deficiency occurring via peroxidase activity, and in Mn deficiency involving peroxidase, Cu-oxidase and fasciclin-like arabinogalactan proteins. Third, the primary cell wall metabolism was affected by both nutrient deficiencies, with changes following opposite directions as judged from the abundances of several glycoside-hydrolases with endo-glycolytic activities and pectin esterases. Fourth, signaling pathways via xylem involving CLE and/or lipids as well as changes in phosphorylation and N-glycosylation also play a role in the responses to these stresses. Biological significance In spite of being essential for the delivery of nutrients to the shoots, our knowledge of xylem responses to nutrient deficiencies is very limited. The present work applies a shotgun proteomic approach to unravel the effects of Fe and Mn deficiencies on the xylem sap proteome. Overall, Fe deficiency seems to elicit more stress in the xylem sap proteome than Mn deficiency, based on the changes measured in proteolytic and oxido-reductase proteins, whereas both nutrients exert modifications in the composition of the primary and secondary cell wall. Cell wall modifications could affect the mechanical and permeability properties of the xylem sap vessels, and therefore ultimately affect solute transport and distribution to the leaves. Results also suggest that signaling cascades involving lipid and peptides might play a role in nutrient stress signaling and pinpoint interesting candidates for future studies. Finally, both nutrient deficiencies seem to affect phosphorylation and glycosylation processes, again following an opposite pattern., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Influenza is a ubiquitous and recurring infection that results in approximately 500 000 deaths globally each year. Commercially available rapid diagnostic tests are based upon detection of the influenza nucleoprotein, which are limited in that they are unable to differentiate by species and require an additional viral lysis step. Sample preprocessing can be minimized or eliminated by targeting the intact influenza virus, thereby reducing assay complexity and leveraging the large number of hemagglutinin proteins on the surface of each virus. Here, we report the development of a paper-based influenza assay that targets the hemagglutinin protein; the assay employs a combination of antibodies and novel computationally designed, recombinant affinity proteins as the capture and detection agents. This system leverages the customizability of recombinant protein design to target the conserved receptor-binding pocket of the hemagglutinin protein and to match the trimeric nature of hemagglutinin for improved avidity. Using this assay, we demonstrate the first instance of intact influenza virus detection using a combination of antibody and affinity proteins within a porous network. The recombinant head region binder based assays yield superior analytical sensitivity as compared to the antibody based assay, with lower limits of detection of 3.54 × 10 7 and 1.34 × 10 7 CEID 50 /mL for the mixed and all binder stacks, respectively. Not only does this work describe the development of a novel influenza assay, it also demonstrates the power of recombinant affinity proteins for use in rapid diagnostic assays.

A prototype of a self-contained, automated, disposable device for chemically amplified protein-based detection of influenza virus from nasal swab specimens was developed and evaluated in a clinical setting. The device required only simple specimen manipulation without any dedicated instrumentation or specialized training by the operator for interpretation. The device was based on a sandwich immunoassay for influenza virus nucleoprotein; it used an enzyme-labeled antibody and a chromogenic substrate to provide an amplified visible signal, in a two-dimensional paper network format. All reagents were stored within the device. Device performance was assessed at Seattle Children's Hospital; clinical staff collected nasal swab samples from 25 patients and then operated test devices on site to detect influenza A and B in those specimens. The total test time from device initiation to result was approximately 35 min. Device performance for influenza A detection was ∼70% accurate using in-house qRT-PCR influenza A as a gold-standard comparison. The ratio of valid to total completed device runs yielded a success rate of 92%, and the negative predictive value for both the influenza A and B assay was 81%. The ability to diagnose respiratory infections rapidly and close to the patient was well received by hospital staff, inspiring further optimization of device function.

Paper microfluidics is a rapidly growing subfield of microfluidics in which paper-like porous materials are used to create analytical devices. There is a need for higher performance field-use tests for many application domains including human disease diagnosis, environmental monitoring, and veterinary medicine. A key factor in creating high performance paper-based devices is the ability to manipulate fluid flow within the devices. This critical review is focused on the progress that has been made in (i) the development of fluid flow control tools and (ii) the integration of those tools into paper microfluidic devices. Further, we strive to be comprehensive in our presentation and provide historical context through discussion and performance comparisons, when possible, of both relevant earlier work and recent work. Finally, we discuss the major areas of focus for fluid flow methods development to advance the potential of paper microfluidics for high-performance field applications.

In the present study we have used label-free shotgun proteomic analysis to examine the effects of Fe deficiency on the protein profiles of highly pure sugar beet root plasma membrane (PM) preparations and detergent-resistant membranes (DRMs), the latter as an approach to study microdomains. Altogether, 545 proteins were detected, with 52 and 68 of them changing significantly with Fe deficiency in PM and DRM, respectively. Functional categorization of these proteins showed that signaling and general and vesicle-related transport accounted for approximately 50% of the differences in both PM and DRM, indicating that from a qualitative point of view changes induced by Fe deficiency are similar in both preparations. Results indicate that Fe deficiency has an impact in phosphorylation processes at the PM level and highlight the involvement of signaling proteins, especially those from the 14-3-3 family. Lipid profiling revealed Fe-deficiency-induced decreases in phosphatidic acid derivatives, which may impair vesicle formation, in agreement with the decreases measured in proteins related to intracellular trafficking and secretion. The modifications induced by Fe deficiency in the relative enrichment of proteins in DRMs revealed the existence of a group of cytoplasmic proteins that appears to be more attached to the PM in conditions of Fe deficiency.

Unlabelled: Iron deficiency is a yield-limiting factor with major implications for crop production, especially in soils with high CaCO3. Because stems are essential for the delivery of nutrients to the shoots, the aim of this work was to study the effects of Fe deficiency on the stem proteome of Medicago truncatula. Two-dimensional electrophoresis separation of stem protein extracts resolved 276 consistent spots in the whole experiment. Iron deficiency in absence or presence of CaCO3 caused significant changes in relative abundance in 10 and 31 spots, respectively, and 80% of them were identified by mass spectrometry. Overall results indicate that Fe deficiency by itself has a mild effect on the stem proteome, whereas Fe deficiency in the presence of CaCO3 has a stronger impact and causes changes in a larger number of proteins, including increases in stress and protein metabolism related proteins not observed in the absence of CaCO3. Both treatments resulted in increases in cell wall related proteins, which were more intense in the presence of CaCO3. The increases induced by Fe-deficiency in the lignin per protein ratio and changes in the lignin monomer composition, assessed by pyrolysis-gas chromatography-mass spectrometry and microscopy, respectively, further support the existence of cell wall alterations., Biological Significance: In spite of being essential for the delivery of nutrients to the shoots, our knowledge of stem responses to nutrient deficiencies is very limited. The present work applies 2-DE techniques to unravel the response of this understudied tissue to Fe deficiency. Proteomics data, complemented with mineral, lignin and microscopy analyses, indicate that stems respond to Fe deficiency by increasing stress and defense related proteins, probably in response of mineral and osmotic unbalances, and eliciting significant changes in cell wall composition. The changes observed are likely to ultimately affect solute transport and distribution to the leaves., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Conventional lateral flow tests (LFTs), the current standard bioassay format used in low-resource point-of-care (POC) settings, have limitations that have held back their application in the testing of low concentration analytes requiring high sensitivity and low limits of detection. LFTs use a premix format for a rapid one-step delivery of premixed sample and labeled antibody to the detection region. We have compared the signal characteristics of two types of reagent delivery formats in a model system of a sandwich immunoassay for malarial protein detection. The premix format produced a uniform binding profile within the detection region. In contrast, decoupling the delivery of sample and labeled antibody to the detection region in a sequential format produced a nonuniform binding profile in which the majority of the signal was localized to the upstream edge of the detection region. The assay response was characterized in both the sequential and premix formats. The sequential format had a 4- to 10-fold lower limit of detection than the premix format, depending on assay conjugate concentration. A mathematical model of the assay quantitatively reproduced the experimental binding profiles for a set of rate constants that were consistent with surface plasmon resonance measurements and absorbance measurements of the experimental multivalent malaria system.

Laboratory-based testing does not allow for the sufficiently rapid return of data to enable optimal therapeutic monitoring of patients with metabolic diseases such as phenylketonuria (PKU). The typical turn-around time of several days for current laboratory-based testing is too slow to be practically useful for effective monitoring or optimizing therapy. This report describes the development of a rapid, paper-based, point-of-care device for phenylalanine detection using a small volume (40 μL) of whole blood. The quantitative resolution and reproducibility of this device with instrumented readout are described, together with the potential use of this device for point-of-care monitoring by PKU patients.

To enable enhanced paper-based diagnostics with improved detection capabilities, new methods are needed to immobilize affinity reagents to porous substrates, especially for capture molecules other than IgG. To this end, we have developed and characterized three novel methods for immobilizing protein-based affinity reagents to nitrocellulose membranes. We have demonstrated these methods using recombinant affinity proteins for the influenza surface protein hemagglutinin, leveraging the customizability of these recombinant "flu binders" for the design of features for immobilization. The three approaches shown are: (1) covalent attachment of thiolated affinity protein to an epoxide-functionalized nitrocellulose membrane, (2) attachment of biotinylated affinity protein through a nitrocellulose-binding streptavidin anchor protein, and (3) fusion of affinity protein to a novel nitrocellulose-binding anchor protein for direct coupling and immobilization. We also characterized the use of direct adsorption for the flu binders, as a point of comparison and motivation for these novel methods. Finally, we demonstrated that these novel methods can provide improved performance to an influenza hemagglutinin assay, compared to a traditional antibody-based capture system. Taken together, this work advances the toolkit available for the development of next-generation paper-based diagnostics.

The aim of this work was to study the effect of Fe deficiency on the protein profile of phloem sap exudates from Brassica napus using 2DE (IEF-SDS-PAGE). The experiment was repeated thrice and two technical replicates per treatment were done. Phloem sap purity was assessed by measuring sugar concentrations. Two hundred sixty-three spots were consistently detected and 15.6% (41) of them showed significant changes in relative abundance (22 decreasing and 19 increasing) as a result of Fe deficiency. Among them, 85% (35 spots), were unambiguously identified. Functional categories containing the largest number of protein species showing changes as a consequence of Fe deficiency were signaling and regulation (32%), and stress and redox homeostasis (17%). The Phloem sap showed a higher oxidative stress and significant changes in the hormonal profile as a result of Fe deficiency. Results indicate that Fe deficiency elicits major changes in signaling pathways involving Ca and hormones, which are generally associated with flowering and developmental processes, causes an alteration in ROS homeostasis processes, and induces decreases in the abundances of proteins involved in sieve element repair, suggesting that Fe-deficient plants may have an impaired capacity to heal sieve elements upon injury., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Failure to utilize valving and automation techniques has restricted the complexity of fluidic operations that can be performed in paper microfluidic devices. We developed a toolkit of paper microfluidic valves and methods for automatic valve actuation using movable paper strips and fluid-triggered expanding elements. To the best of our knowledge, this is the first functional demonstration of this valving strategy in paper microfluidics. After introduction of fluids on devices, valves can actuate automatically after a) a certain period of time, or b) the passage of a certain volume of fluid. Timing of valve actuation can be tuned with greater than 8.5% accuracy by changing lengths of timing wicks, and we present timed on-valves, off-valves, and diversion (channel-switching) valves. The actuators require ~30 μl fluid to actuate and the time required to switch from one state to another ranges from ~5 s for short to ~50 s for longer wicks. For volume-metered actuation, the size of a metering pad can be adjusted to tune actuation volume, and we present two methods - both methods can achieve greater than 9% accuracy. Finally, we demonstrate the use of these valves in a device that conducts a multi-step assay for the detection of the malaria protein PfHRP2. Although slightly more complex than devices that do not have moving parts, this valving and automation toolkit considerably expands the capabilities of paper microfluidic devices. Components of this toolkit can be used to conduct arbitrarily complex, multi-step fluidic operations on paper-based devices, as demonstrated in the malaria assay device.

The fluid collected by direct leaf centrifugation has been used to study the proteome of the sugar beet apoplastic fluid as well as the changes induced by Fe deficiency and Fe resupply to Fe-deficient plants in the protein profile. Plants were grown in Fe-sufficient and Fe-deficient conditions, and Fe resupply was carried out with 45 μM Fe(III)-EDTA for 24 h. Protein extracts of leaf apoplastic fluid were analyzed by two-dimensional isoelectric focusing-SDS-PAGE electrophoresis. Gel image analysis revealed 203 consistent spots, and proteins in 81% of them (164) were identified by nLC-MS/MS using a custom made reference repository of beet protein sequences. When redundant UniProt entries were deleted, a non-redundant leaf apoplastic proteome consisting of 109 proteins was obtained. TargetP and SecretomeP algorithms predicted that 63% of them were secretory proteins. Functional classification of the non-redundant proteins indicated that stress and defense, protein metabolism, cell wall and C metabolism accounted for approximately 75% of the identified proteome. The effects of Fe-deficiency on the leaf apoplast proteome were limited, with only five spots (2.5%) changing in relative abundance, thus suggesting that protein homeostasis in the leaf apoplast fluid is well-maintained upon Fe shortage. The identification of three chitinase isoforms among proteins increasing in relative abundance with Fe-deficiency suggests that one of the few effects of Fe deficiency in the leaf apoplast proteome includes cell wall modifications. Iron resupply to Fe deficient plants changed the relative abundance of 16 spots when compared to either Fe-sufficient or Fe-deficient samples. Proteins identified in these spots can be broadly classified as those responding to Fe-resupply, which included defense and cell wall related proteins, and non-responsive, which are mainly protein metabolism related proteins and whose changes in relative abundance followed the same trend as with Fe-deficiency.

Laboratory-based testing does not reach many individuals in lower-resource settings who could benefit from access to appropriate tests for diagnosis and therapy. A critical issue is laboratory-based testing often requires an environment with a high level of resources and supporting infrastructure that is not available in many areas of the world. The current report describes the conversion of a laboratory-based test for phenylalanine detection to a simple paper-based test appropriate for use in low-resource settings. The paper-based test is easy to operate, with all reagents stored dry on the card, is compatible with visible detection for clinically relevant concentrations of phenylalanine, and has a time to result of 10 minutes. Next steps for test development are discussed in the context of the potential for the paper-based Phe test to be used as a newborn PKU screening test in settings that are not well served by existing screening approaches.

A critical constraint in the design of appropriate medical devices for the lowest-resource settings is the lack of access to maintenance or repair on instrumentation. There are numerous point-of-care applications for which quantitative readout would have clinical utility. Thus, a challenge to the device developer is to enable quantitative device readout in an equipment-free model that is appropriate for use in even the lowest-resource settings. Paper microfluidics has great potential for enabling equipment-free devices that are low-cost, operable by minimally-trained users, and provide quantitative readout. The focus of this critical review is to describe the work, starting several decades ago and continuing to the present, to enable assays with quantitative readout in a fully-disposable device.

Protein profiles of inner (IE) and outer (OE) chloroplast envelope membrane preparations from pea were studied using shotgun nLC-MS/MS and two-dimensional electrophoresis, and 589 protein species (NCBI entries) were identified. The relative enrichment of each protein in the IE/OE pair of membranes was used to provide an integrated picture of the chloroplast envelope. From the 546 proteins identified with shotgun, 321 showed a significant differential distribution, with 180 being enriched in IE and 141 in OE. To avoid redundancy and facilitate in silico localization, Arabidopsis homologues were used to obtain a nonredundant list of 409 envelope proteins, with many showing significant OE or IE enrichment. Functional classification reveals that IE is a selective barrier for transport of many metabolites and plays a major role in controlling protein homeostasis, whereas proteins in OE are more heterogeneous and participate in a wide range of processes. Data support that metabolic processes previously described to occur in the envelope such as chlorophyll and tocopherol biosynthesis can be ascribed to the IE, whereas others such as carotenoid or lipid biosynthesis occur in both membranes. Furthermore, results allow empirical assignation to the IE and/or OE of many proteins previously assigned to the bulk chloroplast envelope proteome.

Lateral flow devices are commonly used for many point-of-care (POC) applications in low-resource settings. However, they lack the sensitivity needed for many analytes relevant in the diagnosis of diseases. One approach to achieve higher sensitivity is signal amplification, which is commonly used in laboratory assays, but uses reagents that require refrigeration and inherently requires multiple assay steps not normally compatible with POC settings. Enzyme-based signal amplification, such as the one used in ELISA, could greatly improve the limit of detection if it were translated to a format compatible with POC requirements. A signal-amplified POC device not only requires the reagents to be stored in a stable form, but also requires automation of the multiple sequential steps of signal amplification protocols. Here, we describe a method for the long-term dry storage of ELISA reagents: horseradish peroxidase (HRP) conjugated antibody label and its colorimetric substrate diaminobenzidine (DAB). The HRP conjugate retained ∼80% enzymatic activity after dry storage at 45 °C for over 5 months. The DAB substrate was also stable at 45 °C and exhibited no detectable loss of activity over 3 months. These reagents were incorporated into a two-dimensional paper network (2DPN) device that automated the steps of ELISA for the detection of a malarial biomarker. These results demonstrate the potential of enzyme-based signal amplification for enhanced sensitivity in POC devices for low resource settings.

Changes induced by three levels of Zn toxicity in the root proteome from Beta vulgaris were studied by two dimensional gel electrophoresis. 320 spots were consistently detected and 5, 5 and 11% of them showed significant changes in relative abundance as a result of the 50, 100 and 300μM Zn treatments, respectively, when compared to controls (1.2μM Zn). Forty-four spots had consistent changes between all treatments, and 93% were identified. At low and mild Zn excess, the complex I of the mitochondrial transport chain and the oxidative phosphorylation were mildly impaired, and an effort to compensate this effect by increasing glycolysis was observed. At high Zn excess, a general metabolism shutdown occurred, as denoted by decreases in the aerobic respiration and by an impairment of the defense systems against oxidative stress. Accordingly, lipid peroxidation increased as Zn supply increased. This study suggests that metabolic changes at high Zn supply reflect cell death, while changes at low and mild Zn supplies may rather explain the metabolic reprogramming occurring upon Zn toxicity. Results also suggest that Zn competition with divalent ions including Fe may contribute to many of the Zn toxicity symptoms, especially at low and moderate Zn supplies., Biological Significance: Results in this work provide a comprehensive overview of the effects of Zn toxicity in roots of sugar beet plants. Effects at low and mild Zn excess are similar and reflect changes in the metabolism aimed to overcome this heavy metal stress, whereas effects at high Zn supply indicate a general shutdown of the metabolism and cell death. Our results indicate that Zn toxicity elicits major impairments in the oxidative stress defense systems, possibly due to Zn competition with divalent cations including Fe, in spite that Zn is not a redox active element by itself., (© 2013 Elsevier B.V. All rights reserved.)

A capability that is key to increasing the performance of paper microfluidic devices is control of fluid transport in the devices. We present dissolvable bridges as a novel method of manipulating fluid volumes within paper-based devices. We demonstrate and characterize the operation of the bridges, including tunability of the volumes passed from 10 μL to 80 μL, using parameters such as geometry and composition. We further demonstrate the utility of dissolvable bridges in the important context of automated delivery of different volumes of a fluid from a common source to multiple locations in a device for simple device loading and activation.

We demonstrate a novel method for controlling fluid flow in paper-based devices. The method delays fluid progress through a porous channel by diverting fluid into an absorbent pad-based shunt placed into contact with the channel. Parameters to control the delay include the length and the thickness of the shunt. Using this method, reproducible delays ranging from 3 to 20 min were achieved. A simple electrical circuit model was presented and used to predict the delays in a system. Results from the model showed good agreement with experimental observations. Finally, the shunts were used for the sequential delivery of fluids to a detection zone in a point-of-care compatible folding card device using biochemical reagents for the amplified detection of the malaria protein PfHRP2.

This Review focuses on recent work in the field of paper microfluidics that specifically addresses the goal of translating the multistep processes that are characteristic of gold-standard laboratory tests to low-resource point-of-care settings. A major challenge is to implement multistep processes with the robust fluid control required to achieve the necessary sensitivity and specificity of a given application in a user-friendly package that minimizes equipment. We review key work in the areas of fluidic controls for automation in paper-based devices, readout methods that minimize dedicated equipment, and power and heating methods that are compatible with low-resource point-of-care settings. We also highlight a focused set of recent applications and discuss future challenges.

Dentists are in a unique position to care for cancer patients before, during and after treatment. This paper outlines foundational knowledge highlighting the dentist's role in the management of patients treated with chemotherapy for various types of cancer as well as those who receive radiation therapy for cancer of the head and neck. The value of the dentist in a multidisciplinary team approach to cancer care is stressed along with specific treatment modalities to improve patient comfort and quality of life.

Lateral flow tests (LFTs) are an ingenious format for rapid and easy-to-use diagnostics, but they are fundamentally limited to assay chemistries that can be reduced to a single chemical step. In contrast, most laboratory diagnostic assays rely on multiple timed steps carried out by a human or a machine. Here, we use dissolvable sugar applied to paper to create programmable flow delays and present a paper network topology that uses these time delays to program automated multi-step fluidic protocols. Solutions of sucrose at different concentrations (10-70% of saturation) were added to paper strips and dried to create fluidic time delays spanning minutes to nearly an hour. A simple folding card format employing sugar delays was shown to automate a four-step fluidic process initiated by a single user activation step (folding the card); this device was used to perform a signal-amplified sandwich immunoassay for a diagnostic biomarker for malaria. The cards are capable of automating multi-step assay protocols normally used in laboratories, but in a rapid, low-cost, and easy-to-use format.

Iron homeostasis is an important process for flower development and plant fertility. The role of plastids in these processes has been shown to be essential. To document the relationships between plastid iron homeostasis and flower biology further, a global study (transcriptome, proteome, metabolome, and hormone analysis) was performed of Arabidopsis flowers from wild-type and triple atfer1-3-4 ferritin mutant plants grown under iron-sufficient or excess conditions. Some major modifications in specific functional categories were consistently observed at these three omic levels, although no significant overlaps of specific transcripts and proteins were detected. These modifications concerned redox reactions and oxidative stress, as well as amino acid and protein catabolism, this latter point being exemplified by an almost 10-fold increase in urea concentration of atfer1-3-4 flowers from plants grown under iron excess conditions. The mutant background caused alterations in Fe-haem redox proteins located in membranes and in hormone-responsive proteins. Specific effects of excess Fe in the mutant included further changes in these categories, supporting the idea that the mutant is facing a more intense Fe/redox stress than the wild type. The mutation and/or excess Fe had a strong impact at the membrane level, as denoted by the changes in the transporter and lipid metabolism categories. In spite of the large number of genes and proteins responsive to hormones found to be regulated in this study, changes in the hormonal balance were restricted to cytokinins, especially in the mutant plants grown under Fe excess conditions.

Novel methods are demonstrated that enable controlled spatial and temporal rehydration of dried reagents in a porous matrix. These methods can be used in paper-based microfluidic assays to define reagent concentrations over time at zones downstream for improved performance, and can reduce costs by simplifying the manufacturing process with the use of a single porous substrate. First, the creation of uniform reagent pulses from patterned arrays of dried reagent is demonstrated. Second, reagents are stored dry in separate regions of the porous matrix so that they can be combined upon rehydration for immediate use in the device. Third, reagents are reconstituted sequentially from dry storage depots with tunable delivery times. Fourth, the total time for dissolution is varied to achieve a range of reagent delivery times to a downstream region. Finally, the utility of these control methods is demonstrated in the context of real-time reagent rehydration and mixing on a porous device.

The lateral flow test has become the standard bioassay format in low-resource settings because it is rapid, easy to use, and low in cost, uses reagents stored in dry form, and is equipment-free. However, lateral flow tests are often limited to a single chemical delivery step and not capable of the multistep processing characteristic of high performance laboratory-based assays. To address this limitation, we are developing a paper network platform that extends the conventional lateral flow test to two dimensions; this allows incorporation of multistep chemical processing, while still retaining the advantages of conventional lateral flow tests. Here, we demonstrate this format for an easy-to-use, signal-amplified sandwich format immunoassay for the malaria protein PfHRP2. The card contains reagents stored in dry form such that the user need only add sample and water. The multiple flows in the device are activated in a single user step of folding the card closed; the configuration of the paper network automatically delivers the appropriate volumes of (i) sample plus antibody conjugated to a gold particle label, (ii) a rinse buffer, and (iii) a signal amplification reagent to the capture region. These results highlight the potential of the paper network platform to enhance access to high-quality diagnostic capabilities in low-resource settings in the developed and developing worlds.

Most laboratory assays take advantage of multi-step protocols to achieve high performance, but conventional paper-based tests (e.g., lateral flow tests) are generally limited to assays that can be carried out in a single fluidic step. We have developed two-dimensional paper networks (2DPNs) that use materials from lateral flow tests but reconfigure them to enable programming of multi-step reagent delivery sequences. The 2DPN uses multiple converging fluid inlets to control the arrival time of each fluid to a detection zone or reaction zone, and it requires a method to disconnect each fluid source in a corresponding timed sequence. Here, we present a method that allows programmed disconnection of fluid sources required for multi-step delivery. A 2DPN with legs of different lengths is inserted into a shared buffer well, and the dropping fluid surface disconnects each leg at in a programmable sequence. This approach could enable multi-step laboratory assays to be converted into simple point-of-care devices that have high performance yet remain easy to use.

Point-of-care diagnostic assays that are rapid, easy-to-use, and low-cost are needed for use in low-resource settings; the lateral flow test has become the standard bioassay format in such settings because it meets those criteria. However, for a number of analytes, conventional lateral flow tests lack the sensitivity needed to have clinical utility. To address this limitation, we are developing a paper network platform that extends the conventional lateral flow test to two dimensions. The two-dimensional structures allow incorporation of multistep processes for improved sensitivity, while still retaining the positive aspects of conventional lateral flow tests. Here we create an easy-to-use, signal-amplified immunoassay based on a modified commercial strip test for human chorionic gonadotropin, the hormone used to detect pregnancy, and demonstrate an improved limit of detection compared to a conventional lateral flow assay. These results highlight the potential of the paper network platform to enhance access to high-quality diagnostic capabilities in low-resource settings in the developed and developing worlds., (© 2011 American Chemical Society)

Two-dimensional paper networks (2DPNs) hold great potential for transcending the capabilities and performance of today's paper-based analytical devices. Specifically, 2DPNs enable sophisticated multi-step chemical processing sequences for sample pretreatment and analysis at a cost and ease-of-use that make them appropriate for use in settings with low resources. A quantitative understanding of flow in paper networks is essential to realizing the potential of these networks. In this report, we provide a framework for understanding flow in simple 2DPNs using experiments, analytical expressions, and computational simulations.

Conventional microfluidic devices typically require highly precise pumps or pneumatic control systems, which add considerable cost and the requirement for power. These restrictions have limited the adoption of microfluidic technologies for point-of-care applications. Paper networks provide an extremely low-cost and pumpless alternative to conventional microfluidic devices by generating fluid transport through capillarity. We revisit well-known microfluidic devices for hydrodynamic focusing, sized-based extraction of molecules from complex mixtures, micromixing, and dilution, and demonstrate that paper-based devices can replace their expensive conventional microfluidic counterparts.

The two-dimensional paper network (2DPN) is a versatile new microfluidic format for performing complex chemical processes. For chemical detection, for example, 2DPNs have the potential to exceed the capabilities and performance of existing paper-based lateral flow devices at a comparable cost and ease of use. To design such 2DPNs, it is necessary to predict 2D flow patterns and velocities within them, but because of the scattering of the paper matrix, conventional particle imaging velocimetry is not practical. In this note, we demonstrate two methods for visualization of flow in 2DPNs that are inexpensive, easy to implement, and quantifiable.