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BACKGROUND:Timely diagnosis of tuberculosis disease is critical for positive patient outcomes, yet potentially millions go undiagnosed or unreported each year. Sputum is widely used as the testing input, but limited by its complexity, heterogeneity, and sourcing problems. Finding methods to interrogate noninvasive, non-sputum clinical specimens is indispensable to improving access to tuberculosis diagnosis and care. In this work, economical plasmonic gratings were used to analyze tuberculosis biomarker lipoarabinomannan (LAM) from clinical urine samples by single molecule fluorescence assay (FLISA) and compared with gold standard sputum GeneXpert MTB/ RIF, culture, and reference ELISA testing results. METHODS AND FINDINGS:In this study, twenty sputum and urine sample sets were selected retrospectively from a repository of HIV-negative patient samples collected before initiation of anti-tuberculosis therapy. GeneXpert MTB/RIF and culture testing of patient sputum confirmed the presence or absence of pulmonary tuberculosis while all patient urines were reference ELISA LAM-negative. Plasmonic gratings produced by low-cost soft lithography were bound with anti-LAM capture antibody, incubated with patient urine samples, and biotinylated detection antibody. Fluorescently labeled streptavidin revealed single molecule emission by epifluorescence microscope. Using a 1 fg/mL baseline for limit of detection, single molecule FLISA demonstrated good qualitative agreement with gold standard tests on 19 of 20 patients, including accurately predicting the gold-standard-negative patients, while one gold-standard-positive patient produced no observable LAM in urine. CONCLUSIONS:Single molecule FLISA by plasmonic grating demonstrated the ability to quantify tuberculosis LAM from complex urine samples of patients from a high endemic setting with negligible interference from the complex media itself. Moreover, agreement with patient diagnoses by gold standard testing suggests that single molecule FLISA could be used as a highly sensitive test to diagnose tuberculosis noninvasively.

Sensitive, accurate, and early detection of biomarkers is essential for prompt response to medical decisions for saving lives. Some infectious diseases are deadly even in small quantities and require early detection for patients and public health. The scarcity of these biomarkers necessitates signal amplification before diagnosis. Recently, we demonstrated single-molecule-level detection of tuberculosis biomarker, lipoarabinomannan, from patient urine using silver plasmonic gratings with thin plasma-activated alumina. While powerful, biomarker binding density was limited by the surface density of plasma-activated carbonyl groups, that degraded quickly, resulting in immediate use requirement after plasma activation. Therefore, development of stable high density binding surfaces such as high binding polystyrene is essential to improving shelf-life, reducing binding protocol complexity, and expanding to a wider range of applications. However, any layers topping the plasmonic grating must be ultra-thin (<10 nm) for the plasmonic enhancement of adjacent signals. Furthermore, fabricating thin polystyrene layers over alumina is nontrivial because of poor adhesion between polystyrene and alumina. Herein, we present the development of a stable, ultra-thin polystyrene layer on the gratings, which demonstrated 63.8 times brighter fluorescence compared to commercial polystyrene wellplates. Spike protein was examined for COVID-19 demonstrating the single-molecule counting capability of the hybrid polystyrene-plasmonic gratings. [ABSTRACT FROM AUTHOR]

The scope of this study was to apply advances in materials science, specifically the use of organosilicate nanoparticles as a high surface area platform for passive sampling of chemicals or pre-concentration for active sensing in multiple-phase complex environmental media. We have developed a novel nanoporous organosilicate (NPO) film as an extraction phase and proof of concept for application in adsorbing hydrophobic compounds in water and sediment. We characterized the NPO film properties and provided optimization for synthesis and coatings in order to apply the technology in environmental media. NPO films in this study had a very high surface area, up to 1325 m2/g due to the high level of mesoporosity in the film. The potential application of the NPO film as a sorbent phase for sensors or passive samplers was evaluated using a model hydrophobic chemical, polychlorinated biphenyls (PCB), in water and sediment. Sorption of PCB to this porous high surface area nanoparticle platform was highly correlated with the bioavailable fraction of PCB measured using whole sediment chemistry, porewater chemistry determined by solid-phase microextraction fiber methods, and the Lumbriculus variegatus bioaccumulation bioassay. The surface-modified NPO films in this study were found to highly sorb chemicals with a log octanol-water partition coefficient (Kow) greater than four; however, surface modification of these particles would be required for application to other chemicals. [ABSTRACT FROM AUTHOR]

Optical sensors have proven to be a useful method in identifying explosive devices by recognizing vapors of explosive compounds that become airborne and emanate from the device. To detect high explosive compounds such as TNT, a molecularly imprinted polymer (MIP) sensing mechanism was developed. This mechanism consists of MIP microparticles prepared using methacrylic acid as the functional monomer. The MIP microparticles are then combined with fluorescent quantum dots via a simple cross-linking procedure. The result is a highly robust optical sensing scheme that is capable of functioning in an array of environmental conditions. To study the sensing mechanisms's ability to detect nitroaromatic analytes, the fluorescent-labeled MIP particles were tested for their performance in detecting aqueous 2,4-dinitrotoluene (DNT), a nitroaromatic molecule very similar to TNT, as well as TNT itself. These preliminary data indicate that the system is capable of detecting nitroaromatic compounds in solution with high sensitivity, achieving lower limits of detection of 30.1 and 40.7 [micro]M for DNT and TNT, respectively. The detection mechanism also acted rapidly, with response times as low as 1 min for TNT. Due to the results of this study, it can be concluded that the fluorescent-labeled MIP system is a feasible method for detecting high explosives, with the potential for future use in detecting vapors from explosive devices. 10.1021/ac902838C

A novel on-chip inexpensive platform to perform DNA amplification has been fabricated by optimizing the design and microfabrication processes using polydimethyl siloxane (PDMS) and silicon. The silicon base contains a set of microfabricated platinum heater structures on the bottom with a 140-nm-thick spin-on-glass (SOG) layer on the top and a 3 [micro]1 replica molded PDMS chamber with feed channels and inlet-outlet ports bonded to this film. The plasma exposed SOG surface is found to undergo recovery of hydrophobicity with time as indicated by an increase in advancing contact angle and bonds very well to another plasma exposed PDMS piece. The bonding protocol developed can be used for a diverse range of substrates, which may form a basis for integration of fluidic assays with microelectronics. The hydrophobic recovery of the microchamber and channels also eliminate the need for various surface passivation techniques for polymerase chain reaction (PCR) chips. A thermal cycler with flexible PCR cycle control is designed and implemented by using a sensing thermocouple. The amplification has been tested using picogram-level template DNA concentration. We have further been able to show negligible nonspecific binding of the template DNA to the hydrophobic interiors of our device by fluorescence measurements and have been able to successfully demonstrate the possibility of multiple usage of this chip without cross-contamination from the previous run. [1708] Index Terms--DNA, infectious bovine rhinetracheitis (IBR), polydimethyl siloxane (PDMS), polymerase chain reaction (PCR) spin-on-glass (SOG), surface recovery.

An issue in microfabrication of the fluidic channels in glass/poly (dimethyl siloxane) (PDMS) is the absence of a well-defined study of the bonding strength between the surfaces making up these channels. Although most of the research papers mention the use of oxygen plasma for developing chemical (siloxane) bonds between the participating surfaces, yet they only define a certain set of parameters, tailored to a specific setup. An important requirement of all the microfluidics/biosensors industry is the development of a general regime, which defines a systematic method of gauging the bond strength between the participating surfaces in advance by correlation to a common parameter. This enhances the reliability of the devices and also gives a structured approach to its future large-scale manufacturing. In this paper, we explore the possibility of the existence of a common scale, which can be used to gauge bond strength between various surfaces. We find that the changes in wettability of surfaces owing to various levels of plasma exposure can be a useful parameter to gauge the bond strength. We obtained a good correlation between contact angle of deionized water (a direct measure of wettability) on the PDMS and glass surfaces based on various dosages of oxygen plasma treatment. The exposure was done first in an inductively coupled high-density (ICP) plasma system and then in plasma enhanced chemical vapor deposition (PECVD) system. This was followed by the measurement of bond strength by use of the standardized blister test. [1336] Index Terms--Advancing contact angle, bond strength, hydrophilicity, hydrophobicity, oxygen-plasma, poly (dimethyl) siloxane (PDMS), reactive ion etching (RIE).