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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.

Rapid and inexpensive serological tests for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antibodies are essential to conduct large-scale seroprevalence surveys and can potentially complement nucleic acid or antigen tests at the point of care. During the COVID-19 pandemic, extreme demand for traditional lateral flow tests has stressed manufacturing capacity and supply chains. Motivated by this limitation, we developed a SARS-CoV-2 antibody test using cellulose, an alternative membrane material, and a double-antigen sandwich format. Functionalized SARS-CoV-2 antigens were used as both capture and reporter binders, replacing the anti-human antibodies currently used in lateral flow tests. The test could provide enhanced sensitivity because it labels only antibodies against SARS-CoV-2 and the signal intensity is not diminished due to other human antibodies in serum. Three-dimensional channels in the assay were designed to have consistent flow rates and be easily manufactured by folding wax-printed paper. We demonstrated that this simple, vertical flow, cellulose-based assay could detect SARS-CoV-2 antibodies in clinical samples within 15 min, and the results were consistent with those from a laboratory, bead-based chemiluminescence immunoassay that was granted emergency use approval by the US FDA.

Oxygen-tolerant radical polymerization has demonstrated applications in biosensors as a signal amplification method for molecular recognition events. In particular, eosin-mediated photo-redox polymerization, a visible light-initiated radical copolymerization method using N-vinyl pyrrolidone and PEDGA monomers, can be performed in aqueous microliter-scale droplets under atmospheric conditions, and has been used for rapid (≤90 s) signal amplification in several diagnostic assays. In recent years, significant progress has been made in understanding the reaction mechanism, and here we assess the accuracy of the proposed mechanism via experimental validation in an assay format. A 2D reaction–diffusion model was developed and compared to experimental behavior of eosin photopolymerization for paper-based signal amplification. For 3 and 4 mm test zones, the model predicted, within an order of magnitude, experimentally observed effects of oxygen exposure and eosin photoinitiator concentration on polymer formation in a droplet. Both model and experimental results demonstrated that high oxygen exposure and low eosin concentration restrict polymer formation to the center of circular wells. Decreasing the surface-area-to-volume ratio of the reaction droplet and increasing eosin concentration allow polymerization throughout the zone, initially forming in radially intermediate zones due to oxygen's role not just as a reaction inhibitor but also a promoter via photoinitiator regeneration. Reaction volumes as low as 20 μL on 3, 4, and 5 mm diameter reaction zones enabled sensitive signal amplification, although higher oxygen exposure (3–10 μL droplets) showed greatly reduced sensitivity. Observing oxygen tolerance limits and experimentally validating the reaction mechanism can help better understand the eosin photopolymerization system and its applications in diagnostic assay signal amplification.

Antibiotics are a vital component of global health. By killing or inhibiting the growth of bacteria, antibiotics treat infections like pneumonia, staph, and tuberculosis. By preventing infections, they enable major medical procedures such as surgeries and chemotherapy. However, bacteria are becoming increasingly resistant to current antibiotics, causing an estimated 34,000 deaths annually in the US. Left unchecked, antibiotic resistance will have major public health consequences, causing over 5 million deaths each year by 2050. Major causes of this crisis are the misuse of existing antibiotics and the slow development of new antibiotics. To incentivize responsible use, governments and institutions are initiating education programs, mandating comprehensive hospital antibiotic stewardship programs, and funding the development of rapid diagnostics. To bring new antibiotic drugs to market, the US government and other non-governmental organizations are funding scientific research toward antibiotic development. Additional incentives are being pursued to improve the commercial viability of antibiotic development and protect drug developers from the unique challenges of the antibiotic market. With diligent efforts to improve responsible use and encourage novel antibiotic drug discovery, we can decrease the global disease burden, save money, and save lives.

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.

The ongoing COVID-19 pandemic has clearly established how vital rapid, widely accessible diagnostic tests are in controlling infectious diseases and how difficult and slow it is to scale existing technologies. Here, we demonstrate the use of the rapid affinity pair identification via directed selection (RAPIDS) method to discover multiple affinity pairs for SARS-CoV-2 nucleocapsid protein (N-protein), a biomarker of COVID-19, from in vitro libraries in 10 weeks. The pair with the highest biomarker sensitivity was then integrated into a 10-minute, vertical-flow cellulose paper test. Notably, the as-identified affinity proteins were compatible with a roll-to-roll printing process for large-scale manufacturing of tests. The test achieved 40 pM and 80 pM limits of detection in 1×PBS (mock swab) and saliva matrices spiked with cell-culture generated SARS-CoV-2 viruses and is also capable of detection of N-protein from characterized clinical swab samples. Hence, this work paves the way towards the mass production of cellulose paper-based assays which can address the shortages faced due to dependence on nitrocellulose and current manufacturing techniques. Further, the results reported herein indicate the promise of RAPIDS and engineered binder proteins for the timely and flexible development of clinically relevant diagnostic tests in response to emerging infectious diseases., Graphical Abstract

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.

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

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 72.4 MJ/m3 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.

Polymerization-based amplification (PBA) uses a visible-light photopolymerization reaction for rapid, low-cost signal amplification and colorimetric detection in disease diagnostics. PBA is particularly useful for point-of-care testing (POCT) in the developing world where laboratory infrastructure and access to advanced technologies may be limited. To date, PBA has required expensive (∼5K USD), desktop-based light sources to drive these photopolymerization reactions, which has limited its use in limited-resource areas and for POCT applications. Here, we present a portable, lightweight, and low-cost device that can generate light of the intensity and wavelengths required to perform the PBA reactions rapidly (

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 83.1 MJ/m3 and from 0.6 to 72.6 MJ/m3 for MSW and mining waste, respectively and was 72.6 MJ/m3 for ash waste. Conductive thermal losses were determined to range from 13 to 1111 MJ/m3 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.