High-Throughput Flow-Through Direct Immunoassays for Targeted Bacteria Detection.

Regulatory authorities require analytical methods for bacteria detection to analyze large sample volumes (typically 100 mL). Currently only the Membrane Filtration and the Most Probable Number assays analyze such large volumes, while other assays for bacteria detection (ELISA, lateral flow assays, etc.) typically analyze volumes 1000 times smaller. This study describes flow-through direct immunoassays (FTDI), a new methodology for the targeted detection of bacteria in liquid samples of theoretically any volume. Flow-through direct immunoassays are performed in fluid-permeable microwells (e.g., wells of a filter well plate) that have a membrane on their bottom where the bacteria are trapped before their detection using a direct immunoassay. Two versions of FTDI assays for the detection of E. coli in 10 mL of sample were developed. A rapid FTDI assay that can be completed in less than 2.5 h can detect E. coli bacteria in levels down to 17 CFU/mL, and an ultrasensitive FTDI assay that employs an additional bacteria culturing step to boost the sensitivity can detect E. coli bacteria in levels lower than 1 CFU/mL in less than 5.5 h. All the steps of the assays, including the immunoassay steps, the culturing step, and the analytical signal measurement step are performed inside the well plate to decrease the chance of contamination and ensure a safe, easy process for the user. The assays were assessed and validated in tap water, river water, and apple juice samples, and the results suggests that the assays are robust, precise, and accurate. When the assays are performed in 96-well filter plates, a filter well plate vacuum manifold and a multichannel peristaltic pump are also used, so multiple samples can be analyzed in parallel to allow high-throughput analysis of samples.

Sink/Float Magnetic Immunoassays for In-Field Bioassays.

Analytical tests/devices that are used outside laboratory settings are required to have a very simple analytical protocol to get clearance by regulatory authorities. This study describes sink/float magnetic immunoassays, a new type of rapid, mix-and-observe, instrument-free tests for the detection of biomarkers in untreated biological samples that are very simple and might meet the simple-to-use criterion of authorities to be used in the field. These tests can tell whether an analyte is above or below a predetermined level within 25-45 minutes based on the sinking or floating of a mm-sized sphere on the surface of which an immunoassay that uses reporter antibodies conjugated to superparamagnetic nanoparticles is performed. This manuscript describes the theory and proof-of-concept applications of sink/float magnetic immunoassays for the detection of C-Reactive Protein, anti-Treponema pallidum antibodies and E. coli bacteria.

Handheld isothermal amplification and electrochemical detection of DNA in resource-limited settings.

This paper demonstrates a new method for electrochemical detection of specific sequences of DNA present in trace amounts in serum or blood. This method is designed for use at the point-of-care (particularly in resource-limited settings). By combining recombinase polymerase amplification (RPA)- an isothermal alternative to the polymerase chain reaction - with an electroactive mediator, this electrochemical methodology enables accurate detection of DNA in the field using a low-cost, portable electrochemical analyzer (specifically designed for this type of analysis). This handheld device has four attributes: (1) It uses disposable, paper-based strips that incorporate screen-printed carbon electrodes; (2) It accomplishes thermoregulation with ±0.1 °C temperature accuracy; (3) It enables electrochemical detection using a variety of pulse sequences, including square-wave and cyclic voltammetry, and coulometry; (4) It is operationally simple to use. Detection of genomic DNA from Mycobacterium smegmatis (a surrogate for M. tuberculosis-the main cause of tuberculosis), and from M. tuberculosis itself down to ∼0.040 ng/µL provides a proof-of-concept for the applicability of this method of screening for disease using molecular diagnostics. With minor modifications to the reagents, this method will also enable field monitoring of food and water quality.

Magnetic Levitation To Characterize the Kinetics of Free-Radical Polymerization.

This work describes the development of magnetic levitation (MagLev) to characterize the kinetics of free-radical polymerization of water-insoluble, low-molecular-weight monomers that show a large change in density upon polymerization. Maglev measures density, and certain classes of monomers show a large change in density when monomers covalently join in polymer chains. MagLev characterized both the thermal polymerization of methacrylate-based monomers and the photopolymerization of methyl methacrylate and made it possible to determine the orders of reaction and the Arrhenius activation energy of polymerization. MagLev also made it possible to monitor polymerization in the presence of solids (aramid fibers, and carbon fibers, and glass fibers). MagLev offers a new analytical technique to materials and polymer scientists that complements other methods (even those based on density, such as dilatometry), and will be useful in investigating polymerizations, evaluating inhibition of polymerizations, and studying polymerization in the presence of included solid materials (e.g., for composite materials).

Universal mobile electrochemical detector designed for use in resource-limited applications.

This paper describes an inexpensive, handheld device that couples the most common forms of electrochemical analysis directly to "the cloud" using any mobile phone, for use in resource-limited settings. The device is designed to operate with a wide range of electrode formats, performs on-board mixing of samples by vibration, and transmits data over voice using audio--an approach that guarantees broad compatibility with any available mobile phone (from low-end phones to smartphones) or cellular network (second, third, and fourth generation). The electrochemical methods that we demonstrate enable quantitative, broadly applicable, and inexpensive sensing with flexibility based on a wide variety of important electroanalytical techniques (chronoamperometry, cyclic voltammetry, differential pulse voltammetry, square wave voltammetry, and potentiometry), each with different uses. Four applications demonstrate the analytical performance of the device: these involve the detection of (i) glucose in the blood for personal health, (ii) trace heavy metals (lead, cadmium, and zinc) in water for in-field environmental monitoring, (iii) sodium in urine for clinical analysis, and (iv) a malarial antigen (Plasmodium falciparum histidine-rich protein 2) for clinical research. The combination of these electrochemical capabilities in an affordable, handheld format that is compatible with any mobile phone or network worldwide guarantees that sophisticated diagnostic testing can be performed by users with a broad spectrum of needs, resources, and levels of technical expertise.

Broadly available imaging devices enable high-quality low-cost photometry.

This paper demonstrates that, for applications in resource-limited environments, expensive microplate spectrophotometers that are used in many central laboratories for parallel measurement of absorbance of samples can be replaced by photometers based on inexpensive and ubiquitous, consumer electronic devices (e.g., scanners and cell-phone cameras). Two devices, (i) a flatbed scanner operating in transmittance mode and (ii) a camera-based photometer (constructed from a cell phone camera, a planar light source, and a cardboard box), demonstrate the concept. These devices illuminate samples in microtiter plates from one side and use the RGB-based imaging sensors of the scanner/camera to measure the light transmitted to the other side. The broadband absorbance of samples (RGB-resolved absorbance) can be calculated using the RGB color values of only three pixels per microwell. Rigorous theoretical analysis establishes a well-defined relationship between the absorbance spectrum of a sample and its corresponding RGB-resolved absorbance. The linearity and precision of measurements performed with these low-cost photometers on different dyes, which absorb across the range of the visible spectrum, and chromogenic products of assays (e.g., enzymatic, ELISA) demonstrate that these low-cost photometers can be used reliably in a broad range of chemical and biochemical analyses. The ability to perform accurate measurements of absorbance on liquid samples, in parallel and at low cost, would enable testing, typically reserved for well-equipped clinics and laboratories, to be performed in circumstances where resources and expertise are limited.

Adaptive use of bubble wrap for storing liquid samples and performing analytical assays.

This paper demonstrates that the gas-filled compartments in the packing material commonly called "bubble wrap" can be repurposed in resource-limited regions as containers to store liquid samples, and to perform bioanalyses. The bubbles of bubble wrap are easily filled by injecting the samples into them using a syringe with a needle or a pipet tip, and then sealing the hole with nail hardener. The bubbles are transparent in the visible range of the spectrum, and can be used as "cuvettes" for absorbance and fluorescence measurements. The interiors of these bubbles are sterile and allow storage of samples without the need for expensive sterilization equipment. The bubbles are also permeable to gases, and can be used to culture and store micro-organisms. By incorporating carbon electrodes, these bubbles can be used as electrochemical cells. This paper demonstrates the capabilities of the bubbles by culturing E. coli, growing C. elegans, measuring glucose and hemoglobin spectrophotometrically, and measuring ferrocyanide electrochemically, all within the bubbles.

Folding analytical devices for electrochemical ELISA in hydrophobic R(H) paper.

This work describes a device for electrochemical enzyme-linked immunosorbent assay (ELISA) designed for low-resource settings and diagnostics at the point of care. The device is fabricated entirely in hydrophobic paper, produced by silanization of paper with decyl trichlorosilane, and comprises two zones separated by a central crease: an embossed microwell, on the surface of which the antigen or antibody immobilization and recognition events occur, and a detection zone where the electrodes are printed. The two zones are brought in contact by folding the device along this central crease; the analytical signal is recorded from the folded configuration. Two proof-of-concept applications, an electrochemical direct ELISA for the detection of rabbit IgG as a model antigen in buffer and an electrochemical sandwich ELISA for the detection of malarial histidine-rich protein from Plasmodium falciparum (Pf HRP2) in spiked human serum, show the versatility of this device. The limit of detection of the electrochemical sandwich ELISA for the quantification of Pf HRP2 in spiked human serum was 4 ng mL(-1) (10(2) pmol L(-1)), a value within the range of clinically relevant concentrations.

The Metabolic and Antioxidant Activity Profiles of Aged Greek Grape Marc Spirits.

In the last decade, "expressions" of grape marc spirits aged in wooden barrels of characteristic amber color and complex sensory attributes have been introduced. Yet studies on constituents migrating from the barrel to the beverage are scarce, and their metabolic profile remains unexplored. Furthermore, the literature on the assessment of their antioxidant activity is limited. NMR metabolomics and spectrophotometry have been implemented in 38 samples to elucidate the impact of the aging procedure on the metabolites' composition and establish whether these beverages exhibit antioxidant activity. Provenance was related to fusel alcohols, esters, acetaldehyde, methanol, saccharides, and 2-phenylethanol, while ethyl acetate and ethyl lactate contributed to discriminating samples of the same winery. Identified metabolites such as vanillin, syringaldehyde, and sinapaldehyde were related to the aging procedure. The maturation in the barrel was also associated with an increase in xylose, glucose, fructose, and arabinose. The antioxidant potential of the aged Greek grape marc spirits resulting from their maturation in oak barrels was highlighted. The metabolic profiling and antioxidant potential of aged Greek grape marc spirits were assessed for the first time. Finally, the enrichment of the aromatic region was noted with the presence of metabolites with a furanic and phenolic ring derived, respectively, from the polysaccharides' degradation or the thermal decomposition of lignin.

Wearable Thermoelectric Devices Based on Three-Dimensional PEDOT:Tosylate/CuI Paper Composites.

Thermoelectric composites of organic and inorganic materials exhibit significantly enhanced thermoelectric properties compared with pristine organic thermoelectrics so they might be better suited as core materials of wearable thermoelectric devices. This study describes the development of three-dimensional (3D) paper PEDOT:tosylate/CuI composites that could be shaped as 3 mm thick blocks to convert a temperature difference between their bottom and top sides into power; the majority of organic thermoelectric materials are shaped as thin strips usually on a planar substrate and convert a temperature difference between the opposite edges of the strips into power. The 3D paper PEDOT:tosylate/CuI composites can produce a power density equal to 4.8 nW/cm2 (ΔΤ = 6 Κ) that is 10 times higher than that of the pristine paper PEDOT:Tos composites. The enhanced thermoelectric properties of the paper PEDOT:tosylate/CuI composites are attributed to the CuI nanocrystals entrapped inside the composite that increases the Seebeck coefficient of the composite to 225 µV K-1; the Seebeck coefficient of paper PEDOT:Tos is 65 µV K-1. A proof-of-concept wearable thermoelectric device that uses 36 blocks of the paper PEDOT:tosylate/CuI composites (as p-type elements) and 36 wires of monel (as n-type elements) can produce up to 4.7 µW of power at ΔΤ = 20 K. The device has a footprint of 64 cm2 and can be placed directly over the skin or can be embedded into clothing.

Inexpensive, Three-Dimensional, Open-Cell, Fluid-Permeable, Noble-Metal Electrodes for Electroanalysis and Electrocatalysis.

This study describes the fabrication of three-dimensional, open-cell, noble-metal (Au, Ag, and Pt) electrodes that have a complex geometry, i.e., wire mesh, metallic foam, "origami" wire mesh, and helix wire mesh. The electrodes were fabricated using an ultrasonication-assisted electroplating method that deposits a thin, continuous, and defect-free layer of noble metal (i.e., Au, Ag, or Pt) on an inexpensive copper substrate that has the desired geometry. The method is inexpensive, easy to use, and capable of fabricating noble-metal electrodes of complex geometries that cannot be fabricated using established techniques like screen printing or physical vapor deposition. By minimizing the amount of the pure noble metal in the electrodes, their cost drops significantly and could become low enough even for single-use applications; for example, the cost of metal in a Au wire-mesh electrode is $0.007/cm2 of exposed area that is about 400 times lower than that of a wire-mesh electrode composed entirely of Au. The electrodes exhibit an almost identical electrochemical performance to noble-metal electrodes of similar shape composed of bulk noble metal; therefore, these electrodes could replace two-dimensional noble-metal electrodes (e.g., rods, disks, foils) in numerous electroanalytical and electrocatalytical systems or even allow the use of noble-metal electrodes in new applications such as flow-based electrochemical systems. In this study, wire-mesh and metallic foam noble-metal electrodes have been successfully used as working electrodes for the electrocatalytical oxidation of methanol and for the electrochemical detection of redox mediators, lead ions, and nitrobenzene using various electroanalytical techniques.

From Point-of-Care Testing to eHealth Diagnostic Devices (eDiagnostics).

Point-of-care devices were originally designed to allow medical testing at or near the point of care by health-care professionals. Some point-of-care devices allow medical self-testing at home but cannot fully cover the growing diagnostic needs of eHealth systems that are under development in many countries. A number of easy-to-use, network-connected diagnostic devices for self-testing are needed to allow remote monitoring of patients' health. This Outlook highlights the essential characteristics of diagnostic devices for eHealth settings and indicates point-of-care technologies that may lead to the development of new devices. It also describes the most representative examples of simple-to-use, point-of-care devices that have been used for analysis of untreated biological samples.

Paper-based devices for biothiols sensing using the photochemical reduction of silver halides.

This study describes the development of paper-based devices for the determination of biothiols. The devices are inexpensive (composed of paper and silver halide particles), and the analytical protocol is easily executable with minimum technical expertise and without the need of specialized equipment; the user has to add a test sample, illuminate the device with a UV lamp, and read the color change of the sensing area using a simple imaging device (i.e., cell-phone camera) or a bare eye. The detection mechanism of the assay is based on the biothiols-mediated photoreduction of nanometer-sized silver chloride particles deposited on the surface of paper; photoreduced silver chloride particles have a grayish coloration that depends on the concentration of biothiols in the tested solution. This is the first time that the UV-mediated photoreduction of solid silver halides particles is used for analytical purposes. The performance of the devices has been tested on the detection of total biothiols content of artificial body fluids and protein-free human blood plasma samples, and the results were satisfactory in terms of sensitivity, selectivity, recoveries and reproducibility.

Determination of phenolic compounds using spectral and color transitions of rhodium nanoparticles.

This work reports a new approach for the determination of phenolic compounds based on their interaction with citrate-capped rhodium nanoparticles. Phenolic compounds (i.e., catechins, gallates, cinnamates, and dihydroxybenzoic acids) were found to cause changes in the size and localized surface plasmon resonance of rhodium nanoparticles, and therefore, give rise to analyte-specific spectral and color transitions in the rhodium nanoparticle suspensions. Upon reaction with phenolic compounds (mainly dithydroxybenzoate derivatives, and trihydroxybenzoate derivatives), new absorbance peaks at 350 nm and 450 nm were observed. Upon reaction with trihydroxybenzoate derivatives, however, an additional absorbance peak at 580 nm was observed facilitating the speciation of phenolic compounds in the sample. Both absorbance peaks at 450 nm and 580 nm increased with increasing concentration of phenolic compounds over a linear range of 0-500 µM. Detection limits at the mid-micromolar levels were achieved, depending on the phenolic compound involved, and with satisfactory reproducibility (<7.3%). On the basis of these findings, two rhodium nanoparticles-based assays for the determination of the total phenolic content and total catechin content were developed and applied in tea samples. The obtained results correlated favorably with commonly used methods (i.e., Folin-Ciocalteu and aluminum complexation assay). Not the least, the finding that rhodium nanoparticles can react with analytes and exhibit unique localized surface plasmon resonance bands in the visible region, can open new opportunities for developing new optical and sensing analytical applications.

Integrating Electronics and Microfluidics on Paper.

Paper microfluidics and printed electronics have developed independently, and are incompatible in many aspects. Monolithic integration of microfluidics and electronics on paper is demonstrated. This integration makes it possible to print 2D and 3D fluidic, electrofluidic, and electrical components on paper, and to fabricate devices using them.

Polymerization-based signal amplification for paper-based immunoassays.

Diagnostic tests in resource-limited settings require technologies that are affordable and easy to use with minimal infrastructure. Colorimetric detection methods that produce results that are readable by eye, without reliance on specialized and expensive equipment, have great utility in these settings. We report a colorimetric method that integrates a paper-based immunoassay with a rapid, visible-light-induced polymerization to provide high visual contrast between a positive and a negative result. Using Plasmodium falciparum histidine-rich protein 2 as an example, we demonstrate that this method allows visual detection of proteins in complex matrices such as human serum and provides quantitative information regarding analyte levels when combined with cellphone-based imaging. It also allows the user to decouple the capture of analyte from signal amplification and visualization steps.

Evaluation of total reducing power of edible oils.

The lipophilicity of untreated edible oils narrows the application of most published methods for the determination of antioxidant activity to hydrophilic extracts of oils. This research addresses the issue of the estimation of the total antioxidant properties of untreated edible oils by modifying two widely applied analytical methods, the Fe-Phenanthroline and the CUPRAC assays, to be used in untreated oils. The modifications pertain to the selection of mixture of solvents (ethanol-butanol in 3:1 v/v ratio), and the optimization of the reaction conditions (reagents concentration and reaction time). The developed methods were applied to a number of hydrophilic and lipophilic standard compounds and different types of commercial edible oils, as well as their corresponding aqueous or organic extracts. This implementation elucidated the differences in the antioxidant content of edible oils. All the results were compared to those of the DPPH and Folin-Ciocalteu methods and the analytical figures of merit for the methods have been estimated. Lastly, it was concluded that the modified CUPRAC assay has higher sensitivity compared to the Fe-Phenanthroline assay.

Development of a generic assay for the determination of total trihydroxybenzoate derivatives based on gold-luminol chemiluminescence.

A selective assay for the determination of one of the most important class of phenolic compounds, namely trihydroxybenzoates (monomeric and polymeric compounds having at least one gallate moiety) based on their enhancing effect on the chemiluminogenic reaction between gold ions and luminol is described for the first time. In the presence of trihydroxybenzoate derivatives, the light emission generated when alkaline luminol is oxidized by gold ions is amplified several orders of magnitude compared to other common phenolic compounds which exhibit minor reactivity or no reactivity at all (e.g. hydroxycinnamates, flavonols, benzenediols). Based on this property, the experimental conditions were optimized in order to enable the determination of total trihydroxybenzoates in complex mixtures without resorting to separation techniques. The method was applied to samples of different composition (teas, herbal infusions and wines) with satisfactory analytical features yielding detection limits at the 10(-7) mol L(-1) level, intra-day precision of 3.1%, inter-day precision less than 10% and recoveries between 88.7 and 97.6%. The strengths and weaknesses of the method were identified and discussed in relation to its application in real samples.