The Quantitative Detection of Cystatin-C in Patient Samples Using a Colorimetric Lateral Flow Immunoassay.

A colloidal gold-based lateral flow immunoassay was developed for the rapid quantitative detection of Cystatin-C in serum and whole blood. This device has an assay time of 15 min, making it a convenient point-of-care diagnostic tool. The device has a quantification range spanning from 0.5 to 7.5 µg/mL, with a lower limit of detection at 0.18 µg/mL. To validate its accuracy, the test was compared to a standard nephelometric immunoassay, and the results exhibited a robust linear correlation with an adjusted r2 value of 0.95. Furthermore, the device demonstrates satisfactory levels of analytical performance in terms of precision, sensitivity, and interference, indicating its potential for precise Cystatin-C quantification, particularly in renal-failure patients. Notably, the Cystatin-C-LFA device also demonstrates satisfactory stability, as a 30-day accelerated stability study at 50 °C showed no change in the device performance, indicating a long shelf life for the product when stored at room temperature.

Microbial Community Distribution and Core Microbiome in Successive Wound Grades of Individuals with Diabetic Foot Ulcers.

Diabetic foot ulcer (DFU) is a major complication of diabetes with high morbidity and mortality rates. The pathogenesis of DFUs is governed by a complex milieu of environmental and host factors. The empirical treatment is initially based on wound severity since culturing and profiling the antibiotic sensitivity of wound-associated microbes is time-consuming. Hence, a thorough and rapid analysis of the microbial landscape is a major requirement toward devising evidence-based interventions. Toward this, 122 wound (100 diabetic and 22 nondiabetic) samples were sampled for their bacterial community structure using both culture-based and next-generation 16S rRNA-based metagenomics approach. Both the approaches showed that the Gram-negative microbes were more abundant in the wound microbiome. The core microbiome consisted of bacterial genera, including Alcaligenes, Pseudomonas, Burkholderia, and Corynebacterium in decreasing order of average relative abundance. Despite the heterogenous nature and extensive sharing of microbes, an inherent community structure was apparent, as revealed by a cluster analysis based on Euclidean distances. Facultative anaerobes (26.5%) were predominant in Wagner grade 5, while strict anaerobes were abundant in Wagner grade 1 (26%). A nonmetric dimensional scaling analysis could not clearly discriminate samples based on HbA1c levels. Sequencing approach revealed the presence of major culturable species even in samples with no bacterial growth in culture-based approach. Our study indicates that (i) the composition of core microbial community varies with wound severity, (ii) polymicrobial species distribution is individual specific, and (iii) antibiotic susceptibility varies with individuals. Our study suggests the need to evolve better-personalized care for better wound management therapies.IMPORTANCE Chronic nonhealing diabetic foot ulcers (DFUs) are a serious complication of diabetes and are further exacerbated by bacterial colonization. The microbial burden in the wound of each individual displays diverse morphological and physiological characteristics with unique patterns of host-pathogen interactions, antibiotic resistance, and virulence. Treatment involves empirical decisions until definitive results on the causative wound pathogens and their antibiotic susceptibility profiles are available. Hence, there is a need for rapid and accurate detection of these polymicrobial communities for effective wound management. Deciphering microbial communities will aid clinicians to tailor their treatment specifically to the microbes prevalent in the DFU at the time of assessment. This may reduce DFUs associated morbidity and mortality while impeding the rise of multidrug-resistant microbes.

Design of Hydrogel Particle Morphology for Rapid Bioassays.

Hydrogel microparticles have been extensively used in the field of medical diagnostics for detecting targets ranging from proteins to nucleic acids. However, little is known about how the shape of hydrogel particles impacts the signal from a bioassay. In this article, we analyze the flux into porous hydrogel particles to develop scaling laws for the signal from a point-of-care bioassay. The signal can be increased by increasing the ratio of the surface area of the hydrogel particle to the two-dimensional projected imaging area used for analysis. We show that adding internal surface area to hydrogel particles increases the assay signal in a biotin-streptavidin bioassay. We also demonstrate the application of this technique to a protein-based assay for thyroid-stimulating hormone, reducing the limit of detection of the assay sixfold by changing particle shape. We anticipate that these strategies can be used broadly to optimize hydrogel-based systems for point-of-care diagnostics.

Electrically-Actuated Valves for Woven Fabric Lateral Flow Devices.

The integration of flow control elements into low-cost biosensors presents a significant engineering challenge. This Article describes the development and integration of active, chemical valves into lateral flow devices, using a scalable, single-step, weaving-based manufacturing approach. The valve was constructed from an electrically conductive polymer, polypyrrole. The polymer switches between wetting and nonwetting states when it is reduced and oxidized via the application of an electrochemical potential. In this work, yarns were first coated with polypyrrole and integrated into fabric lateral flow sensors. The coated yarns were stimulated in situ via integrated electrodes. Coated textiles were characterized for their response to variations in the applied electrical potential, the duration for which the potential is applied, and the chemical composition of the polymer. Among these tuning parameters, the concentration of iron (iii) chloride utilized to catalyze the synthesis of the polymer, was found to be a significant determinant in the wetting range of the polymer. Complete ON/OFF flow control was achieved at applied potentials of 20 V.cm-1, within 120 s of stimulation, using 0.1 M iron (iii) chloride, making the valve fairly easy to incorporate into point-of-care format. The practical utility of the valve was demonstrated by performing a Lowry protein assay in the device, wherein fluid flow was deactivated to allow individual reaction steps to go to completion prior to reactivation. Significant improvements in the sensitivity and linear range of the devices are reported in a simple straight-channel, lateral flow device, with the potential to develop more complex channel geometries via the weaving-based approach.

Automated ABO Rh-D blood type detection using smartphone imaging for point-of-care medical diagnostics.

We present a novel methodology for automated ABO Rh-D blood typing using simple morphological image processing algorithms to be used in conjunction with a fabric strip based rapid diagnostic test. Images of the fabric strip post testing are acquired using low cost mobile phones and the proposed algorithm proceeds to automatically identify the blood type by processing the images using steps comprising of noise reduction, range filtering and empirically derived heuristics. The ultimate goal is to provide a simple mobile phone application to enable automated, rapid and accessible blood type detection at the point-of-care.

Woven electrochemical fabric-based test sensors (WEFTS): a new class of multiplexed electrochemical sensors.

We present textile weaving as a new technique for the manufacture of miniature electrochemical sensors with significant advantages over current fabrication techniques. Biocompatible silk yarn is used as the material for fabrication instead of plastics and ceramics used in commercial sensors. Silk yarns are coated with conducting inks and reagents before being handloom-woven as electrodes into patches of fabric to create arrays of sensors, which are then laminated, cut and packaged into individual sensors. Unlike the conventionally used screen-printing, which results in wastage of reagents, yarn coating uses only as much reagent and ink as required. Hydrophilic and hydrophobic yarns are used for patterning so that sample flow is restricted to a small area of the sensor. This simple fluidic control is achieved with readily available materials. We have fabricated and validated individual sensors for glucose and hemoglobin and a multiplexed sensor, which can detect both analytes. Chronoamperometry and differential pulse voltammetry (DPV) were used to detect glucose and hemoglobin, respectively. Industrial quantities of these sensors can be fabricated at distributed locations in the developing world using existing skills and manufacturing facilities. We believe such sensors could find applications in the emerging area of wearable sensors for chemical testing.

Tunable electrophoretic separations using a scalable, fabric-based platform.

There is a rising need for low-cost and scalable platforms for sensitive medical diagnostic testing. Fabric weaving is a mature, scalable manufacturing technology and can be used as a platform to manufacture microfluidic diagnostic tests with controlled, tunable flow. Given its scalability, low manufacturing cost (<$0.25 per device), and potential for patterning multiplexed channel geometries, fabric is a viable platform for the development of analytical devices. In this paper, we describe a fabric-based electrophoretic platform for protein separation. Appropriate yarns were selected for each region of the device and weaved into straight channel electrophoretic chips in a single step. A wide dynamic range of analyte molecules ranging from small molecule dyes (<1 kDa) to macromolecule proteins (67-150 kDa) were separated in the device. Individual yarns behave as a chromatographic medium for electrophoresis. We therefore explored the effect of yarn and fabric parameters on separation resolution. Separation speed and resolution were enhanced by increasing the number of yarns per unit area of fabric and decreasing yarn hydrophilicity. However, for protein analytes that often require hydrophilic, passivated surfaces, these effects need to be properly tuned to achieve well-resolved separations. A fabric device tuned for protein separations was built and demonstrated. As an analytical output parameter for this device, the electrophoretic mobility of a sedimentation marker, Naphthol Blue Black bovine albumin in glycine-NaOH buffer, pH 8.58 was estimated and found to be -2.7 × 10(-8) m(2) V(-1) s(-1). The ability to tune separation may be used to predefine regions in the fabric for successive preconcentrations and separations. The device may then be applied for the multiplexed detection of low abundance proteins from complex biological samples such as serum and cell lysate.

The development of "fab-chips" as low-cost, sensitive surface-enhanced Raman spectroscopy (SERS) substrates for analytical applications.

The demand for methods and technologies capable of rapid, inexpensive and continuous monitoring of health status or exposure to environmental pollutants persists. In this work, the development of novel surface-enhanced Raman spectroscopy (SERS) substrates from metal-coated silk fabric, known as zari, presents the potential for SERS substrates to be incorporated into clothing and other textiles for the routine monitoring of important analytes, such as disease biomarkers or environmental pollutants. Characterization of the zari fabric was completed using scanning electron microscopy, energy dispersive X-ray analysis and Raman spectroscopy. Silver nanoparticles (AgNPs) were prepared, characterized by transmission electron microscopy and UV-vis spectroscopy, and used to treat fabric samples by incubation, drop-coating and in situ synthesis. The quality of the treated fabric was evaluated by collecting the SERS signal of 4,4'-bipyridine on these substrates. When AgNPs were drop-coated on the fabric, sensitive and reproducible substrates were obtained. Adenine was selected as a second probe molecule, because it dominates the SERS signal of DNA, which is an important class of disease biomarker, particularly for pathogens such as Plasmodium spp. and Mycobacterium tuberculosis. Excellent signal enhancement could be achieved on these affordable substrates, suggesting that the developed fabric chips have the potential for expanding the use of SERS as a diagnostic and environmental monitoring tool for application in wearable sensor technologies.

'Fab-chips': a versatile, fabric-based platform for low-cost, rapid and multiplexed diagnostics.

Low cost and scalable manufacture of lab-on-chip devices for applications such as point-of-care testing is an urgent need. Weaving is presented as a unified, scalable and low-cost platform for the manufacture of fabric chips that can be used to perform such testing. Silk yarns with different properties are first selected, treated with the appropriate reagent solutions, dried and handloom-woven in one step into an integrated fabric chip. This platform has the unique advantage of scaling up production using existing and low cost physical infrastructure. We have demonstrated the ability to create pre-defined flow paths in fabric by using wetting and non-wetting silk yarns and a Jacquard attachment in the loom. Further, we show that yarn parameters such as the yarn twist frequency and weaving coverage area may be conveniently used to tune both the wicking rate and the absorptive capacity of the fabric. Yarns optimized for their final function were used to create an integrated fabric chip containing reagent-coated yarns. Strips of this fabric were then used to perform a proof-of-concept immunoassay with sample flow taking place by capillary action and detection being performed by a visual readout.