Accelerating the optimization of vertical flow assay performance guided by a rational systematic model-based approach.

Rapid diagnostic tests (RDTs) have shown to be instrumental in healthcare and disease control. However, they have been plagued by many inefficiencies in the laborious empirical development and optimization process for the attainment of clinically relevant sensitivity. While various studies have sought to model paper-based RDTs, most have relied on continuum-based models that are not necessarily applicable to all operation regimes, and have solely focused on predicting the specific interactions between the antigen and binders. It is also unclear how the model predictions may be utilized for optimizing assay performance. Here, we propose a streamlined and simplified model-based framework, only relying on calibration with a minimal experimental dataset, for the acceleration of assay optimization. We show that our models are capable of recapitulating experimental data across different formats and antigen-binder-matrix combinations. By predicting signals due to both specific and background interactions, our facile approach enables the estimation of several pertinent assay performance metrics such as limit-of-detection, sensitivity, signal-to-noise ratio and difference. We believe that our proposed workflow would be a valuable addition to the toolset of any assay developer, regardless of the amount of resources they have in their arsenal, and aid assay optimization at any stage in their assay development process.

Polymerization-Based Amplification for Target-Specific Colorimetric Detection of Amplified Mycobacterium tuberculosis DNA on Cellulose.

Loop-mediated isothermal amplification (LAMP) is an appealing method for low-cost, point-of-care nucleic acid diagnostic assays due to high sensitivity, minimal equipment requirements, and compatibility with user-friendly colorimetric detection methods. The enhanced sensitivity LAMP offers comes with vulnerability to cross-contamination, where negative samples are exposed to minute amounts of nucleic acids from positive samples. These amounts are insignificant in less sensitive amplification methods, but visible when LAMP is paired with common colorimetric methods. Here, we examined the use of eosin photopolymerization, a tunable reaction, for colorimetric detection of LAMP products to reduce this false positive risk. Using eosin and biotin end-labeled primers, we successfully amplified target regions of the Mycobacterium tuberculosis (MTB) genome using PCR and LAMP, captured amplicons on streptavidin-coated cellulose, and detected DNA targets via eosin photopolymerization, producing a bright pink color only if MTB DNA was present in the sample. Consistent with previous reports, the LAMP-based method exhibited high background signal, but tuning the illumination time for the photopolymerization reaction allowed readouts from samples with no added MTB DNA to remain blank and visually distinct from pink positives. This method yielded limits of detection of 30 and 300 copies/µL for LAMP and PCR amplification, respectively. When confronted with boiled MTB culture samples, this method gave clear positive readouts, compared to negligible signal from other Mycobacterium boiled culture samples. This new method of LAMP colorimetric detection has the potential to increase the utility of LAMP as a nucleic acid assay technique by mitigating sensitivity to cross-contamination.

Detection of Biomarkers of Periodontal Disease in Human Saliva Using Stabilized, Vertical Flow Immunoassays.

We report methods for stabilizing cellulose-based immunoassays and using this platform to analyze human saliva. Stabilization treatments of immunoassays for matrix metalloproteinases (MMP)-8 and -9, biomarkers of periodontal disease, were conducted and compared, revealing that anti-MMP-8 and -9 capture antibodies could be stabilized with the addition of a 5% trehalose solution to the test zones, followed by drying in a vacuum oven. After stabilization, the paper devices retained equivalent binding activity to that of freshly prepared tests for 14 days-a time frame that enables US-based clinical testing of this diagnostic assay. A saliva pretreatment method was developed to remove viscous elements without reducing the concentration or binding activity of dissolved proteins. Immunoassays were stored in ziplock bags containing desiccant, and used to detect nanomolar concentrations of MMP-9 in human saliva across the relevant clinical concentration range. These methods and findings facilitate rapid, affordable validation studies of this and other biomarkers that are found in saliva using vertical flow immunoassays.

Heat management strategies for MSW landfills.

Heat is a primary byproduct of landfilling of municipal solid waste. Long-term elevated temperatures have been reported for MSW landfills under different operational conditions and climatic regions around the world. A conceptual framework is presented for management of the heat generated in MSW landfills. Three main strategies are outlined: extraction, regulation, and supplementation. Heat extraction allows for beneficial use of the excess landfill heat as an alternative energy source. Two approaches are provided for the extraction strategy: extracting all of the excess heat above baseline equilibrium conditions in a landfill and extracting only a part of the excess heat above equilibrium conditions to obtain target optimum waste temperatures for maximum gas generation. Heat regulation allows for controlling the waste temperatures to achieve uniform distribution at target levels at a landfill facility. Two approaches are provided for the regulation strategy: redistributing the excess heat across a landfill to obtain uniform target optimum waste temperatures for maximum gas generation and redistributing the excess heat across a landfill to obtain specific target temperatures. Heat supplementation allows for controlling heat generation using external thermal energy sources to achieve target waste temperatures. Two approaches are provided for the supplementation strategy: adding heat to the waste mass using an external energy source to increase waste temperatures and cooling the waste mass using an external energy source to decrease waste temperatures. For all strategies, available landfill heat energy is determined based on the difference between the waste temperatures and the target temperatures. Example analyses using data from landfill facilities with relatively low and high heat generation indicated thermal energy in the range of -48.4 to 72.4MJ/m(3) available for heat management. Further modeling and experimental analyses are needed to verify the effectiveness and feasibility of design, installation, and operation of heat management systems in MSW landfills.

Waste heat generation: A comprehensive review.

A comprehensive review of heat generation in various types of wastes and of the thermal regime of waste containment facilities is provided in this paper. Municipal solid waste (MSW), MSW incineration ash, and mining wastes were included in the analysis. Spatial and temporal variations of waste temperatures, thermal gradients, thermal properties of wastes, average temperature differentials, and heat generation values are provided. Heat generation was influenced by climatic conditions, mean annual earth temperatures, waste temperatures at the time of placement, cover conditions, and inherent heat generation potential of the specific wastes. Time to onset of heat generation varied between months and years, whereas timelines for overall duration of heat generation varied between years and decades. For MSW, measured waste temperatures were as high as 60-90°C and as low as -6°C. MSW incinerator ash temperatures varied between 5 and 87°C. Mining waste temperatures were in the range of -25 to 65°C. In the wastes analyzed, upward heat flow toward the surface was more prominent than downward heat flow toward the subsurface. Thermal gradients generally were higher for MSW and incinerator ash and lower for mining waste. Based on thermal properties, MSW had insulative qualities (low thermal conductivity), while mining wastes typically were relatively conductive (high thermal conductivity) with ash having intermediate qualities. Heat generation values ranged from -8.6 to 83.1MJ/m(3) and from 0.6 to 72.6MJ/m(3) for MSW and mining waste, respectively and was 72.6MJ/m(3) for ash waste. Conductive thermal losses were determined to range from 13 to 1111MJ/m(3)yr. The data and analysis provided in this review paper can be used in the investigation of heat generation and thermal regime of a wide range of wastes and waste containment facilities located in different climatic regions.