Sensitive, Single-Pot, Two-Stage Assay for Hepatitis Viruses.

When left untreated, hepatitis B virus (HBV) and hepatitis C virus (HCV) infections may cause severe illnesses. Since these infections remain asymptomatic for many years, routine screening of populations at risk is critical for therapy initiation. The current standard of care mandates a screening antibody test for HCV, followed by a confirmatory laboratory-based molecular test and treatment. Multiple visits to the clinic are inconvenient, and many patients fail to follow up. To address this challenge, we have developed sensitive, two-stage, isothermal molecular (Penn-RAMP) point-of-care tests to enable test and treat strategy. Penn-RAMP's first stage is comprised of recombinase polymerase amplification (RPA), while its second stage is comprised of loop-mediated isothermal amplification (LAMP). Penn-RAMP is more sensitive than LAMP or RPA alone. We designed a custom pre-LAMP buffer to maximize the volume of RPA products that can be added to the LAMP reaction mix without inhibition and forward and backward primers. Penn-RAMP was implemented in a single pot comprised of two compartments separated by a thermally removable barrier. RAMP's first stage is carried out above the barrier at the RPA incubation temperature. When the pot is heated to the LAMP incubation temperature, the barrier melts away, and the RPA reaction volume mixes with the pre-LAMP buffer, facilitating second-stage amplification. This entire process can be carried out with minimal instrumentation. Our HBV and HCV tests detect, respectively, as few as 10 and 25 virions within 30 min. The viral load can be estimated based on signal threshold time.

Manually-Operated, Slider Cassette for Multiplexed Molecular Detection at the Point of Care.

Effective control of epidemics, individualized medicine, and new drugs with virologic response-dependent dose and timing require, among other things, simple, inexpensive, multiplexed molecular detection platforms suitable for point of care and home use. Herein, we describe our progress towards developing such a platform that includes sample lysis, nucleic acid isolation, concentration, purification, and amplification. Our diagnostic device comprises a sliding component that houses the nucleic acid isolation membrane and a housing containing three amplification reaction chambers with dry stored reagents, blisters with buffers and wash solutions, and absorption pads to facilitate capillarity pull and waste storage. After sample introduction, the user slides the slider within the housing from one station to another to carry out various unit operations. The slider motion induces blisters to discharge their contents, effectuating washes, and eventual elution of captured nucleic acids into reaction chambers. The slider cassette mates with a processor that incubates isothermal amplification but can also be made to operate instrumentation-free. We demonstrate our cassette's utility for the co-detection of the human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV). These three blood-borne pathogens co-infect many people worldwide with severe personal and public health consequences.

A Size-Selectively Biomolecule-Immobilized Nanoprobe-Based Chemiluminescent Lateral Flow Immunoassay for Detection of Avian-Origin Viruses.

In this study, a signal-amplifiable nanoprobe-based chemiluminescent lateral flow immunoassay (CL-LFA) was developed to detect avian influenza viruses (AIV) and other contagious and fatal viral avian-origin diseases worldwide. Signal-amplifiable nanoprobes are capable of size-selective immobilization of antibodies (binding receptors) and enzymes (signal transducers) on sensitive paper-based sensor platforms. Particle structure designs and conjugation pathways conducive for antigen accessibility to maximum amounts of immobilized enzymes and antibodies have advanced. The detection limit of the CL-LFA using the signal-amplifiable nanoprobe for the nucleoprotein of the H3N2 virus was 5 pM. Sensitivity tests for low pathogenicity avian influenza H9N2, H1N1, and high pathogenicity avian influenza H5N9 viruses were conducted, and the detection limits of CL-LFA were found to be 103.5 50% egg infective dose (EID50)/mL, 102.5 EID50/mL, and 104 EID50/mL, respectively, which is 20 to 100 times lower than that of a commercial AIV rapid test kit. Moreover, CL-LFA demonstrated high sensitivity and specificity against 37 clinical samples. The signal-amplifiable probe designed in this study is a potential diagnostic probe with ultrahigh sensitivity for applications in the field of clinical diagnosis, which requires sensitive antigen detection as evidenced by enhanced signaling capacity and sensitivity of the LFAs.

Electricity-free chemical heater for isothermal nucleic acid amplification with applications in COVID-19 home testing.

Molecular detection of pathogenic nucleic acids from patient samples requires incubating biochemical reactions at specific temperatures to amplify DNA. This incubation is typically carried out with an electrical heater and a temperature controller. To reduce test cost, to eliminate the need for manufacturing incubators, which may require significant time, and to enable electricity-free operation, we use energetic compounds such as an Mg(Fe) alloy mixed with a phase-change material (PCM) that undergoes phase transformation at the desired incubation temperature. We dubbed this composite Energetic Phase Change Material (EPCM). When the EPCM is brought into contact with water, the magnesium alloy interacts with the water to produce heat. The EPCM heats up to its phase transition temperature. Any excess heat is absorbed as latent heat and the system is maintained at its desired incubation temperature, independent of ambient temperatures, long enough to facilitate enzymatic amplification. The EPCM together with colorimetric amplicon detection facilitates an inexpensive, disposable, point-of-need diagnostic test that does not require any electric power. We demonstrate the feasibility of our approach by detecting SARS-Cov-2 in saliva samples either without any instrumentation or with a palm-size CCD camera that enables us to follow the amplification process in real time.

Single-Step Recombinase Polymerase Amplification Assay Based on a Paper Chip for Simultaneous Detection of Multiple Foodborne Pathogens.

A paper chip device-based recombinase polymerase amplification (RPA) method was developed for highly sensitive and selective single-step detection of foodborne pathogens. A paper chip was manufactured by simply stacking functional papers. RPA reagents and fluorescent probe were dried on the reaction zone of a patterned poly(ether sulfone) membrane. The RPA reaction was initiated by adding pathogen DNAs into an injection hole. Paper chip-based analysis of pathogens showed optimal performance at 37 °C for 20 min and the results were comparable to those obtained with solution-based RPA reactions. Based on the paper chip-based fluorescence signal, Escherichia coli, Staphylococcus aureus, and Salmonella typhimurium were simultaneously detected with detection limits of 102 cfu/mL. The diagnostic utility of the device was demonstrated by the reliable detection of E. coli and S. aureus present in spiked milk. This ready-to-use device could be integrated with simple nucleic acid extraction for food pathogen detection in resource-limited settings.

Trends of respiratory virus detection in point-of-care testing: A review.

In resource-limited conditions such as the COVID-19 pandemic, on-site detection of diseases using the Point-of-care testing (POCT) technique is becoming a key factor in overcoming crises and saving lives. For practical POCT in the field, affordable, sensitive, and rapid medical testing should be performed on simple and portable platforms, instead of laboratory facilities. In this review, we introduce recent approaches to the detection of respiratory virus targets, analysis trends, and prospects. Respiratory viruses occur everywhere and are one of the most common and widely spreading infectious diseases in the human global society. Seasonal influenza, avian influenza, coronavirus, and COVID-19 are examples of such diseases. On-site detection and POCT for respiratory viruses are state-of-the-art technologies in this field and are commercially valuable global healthcare topics. Cutting-edge POCT techniques have focused on the detection of respiratory viruses for early diagnosis, prevention, and monitoring to protect against the spread of COVID-19. In particular, we highlight the application of sensing techniques to each platform to reveal the challenges of the development stage. Recent POCT approaches have been summarized in terms of principle, sensitivity, analysis time, and convenience for field applications. Based on the analysis of current states, we also suggest the remaining challenges and prospects for the use of the POCT technique for respiratory virus detection to improve our protection ability and prevent the next pandemic.

Paper-Based Airborne Bacteria Collection and DNA Extraction Kit.

The critical risk from airborne infectious diseases, bio-weapons, and harmful bacteria is currently the highest it has ever been in human history. The requirement for monitoring airborne pathogens has gradually increased to defend against bioterrorism or prevent pandemics, especially via simple and low-cost platforms which can be applied in resource-limited settings. Here, we developed a paper-based airborne bacteria collection and DNA extraction kit suitable for simple application with minimal instruments. Airborne sample collection and DNA extraction for PCR analysis were integrated in the paper kit. We created an easy-to-use paper-based air monitoring system using 3D printing technology combined with an air pump. The operation time of the entire process, comprising air sampling, bacterial cell lysis, purification and concentration of DNA, and elution of the DNA analyte, was within 20 min. All the investigations and optimum settings were tested in a custom-designed closed cabinet system. In the fabricated cabinet system, the paper kit operated effectively at a temperature of 25-35 °C and 30-70% relative humidity for air containing 10-106 CFU Staphylococcus aureus. This paper kit could be applied for simple, rapid, and cost-effective airborne pathogen monitoring.

Lab-on-paper for all-in-one molecular diagnostics (LAMDA) of zika, dengue, and chikungunya virus from human serum.

Several tropical fever viruses transmitted by mosquitoes including zika, dengue, and chikungunya, are becoming a serious problem in global public health. Simple diagnostic tools in early stages are strongly required to monitor and prevent these diseases. Paper diagnostic platforms can provide a solution for these needs, with integration of fluidic control techniques and isothermal amplification methods. Here, we demonstrate a Lab-on-paper for all-in-one molecular diagnostics of zika, dengue, and chikungunya virus from human serum. The entire process of nucleic acid testing that involves sampling, extraction, amplification, and detection is simply operated on a single paper chip. Based on the engineered structure of paper materials and dried chemicals on the all-in-one chip, serum samples containing the target virus RNA were simply added by automatic flow from distilled water injection. Target RNA molecules were concentrated on the binding pad with chitosan and then transported to reaction pads following a pH increase for specific reverse transcription loop-mediated isothermal amplification with fluorescence signal generation. Three targets, zika virus, dengue virus, and chikungunya virus, in human serum were simultaneously detected on the all-in-one paper chip within 60 min at 65 °C. The all-in-one paper chip can be used as a real-time quantitative assay for 5-5000 copies of zika virus RNA. This all-in-one device was successfully used with 5 clinical specimens of zika and dengue virus from real patients. We believe that the proposed all-in-one paper chip can provide a portable, low-cost, user-friendly, sensitive, and specific NAT platform with great potential in point-of-care diagnostics.

An innovative paper-based device for DNA extraction from processed meat products.

Detection of food adulteration is a challenge. However, the identification of adulterated meat in processed products is important for health and personal preference. Mitochondrial genomic DNA (mtDNA) is a good candidate for reliable identification of meat ingredients; however, the extraction of mtDNA from processed products is a bottleneck for development of detection strategies. Therefore, we constructed a rapid (~5 min) mtDNA extraction device. mtDNAs from different meat samples, such as pork (Sus scrofa), chicken (Gallus gallus), and beef (Bos taurus), were successfully detected in up to 0.1% adulterated animal species. We believe that the proposed strategy could be applied to detect animal species from processed meat products to reduce fraudulent practices.

Paper-based nucleic acid testing system for simple and early diagnosis of mosquito-borne RNA viruses from human serum.

The recent outbreaks of mosquito-borne diseases (e.g., zika, dengue, and chikungunya) increased public health burden in developing countries. To control the spread of these infectious diseases, a simple, economic, reliable, sensitive, and selective diagnostic platform is required. Considering demand for affordable and accessible methods, we have demonstrated a two-step strategy for extraction and detection of viral RNAs of infectious diseases within 1 h. Ready-to-use devices for viral RNA extraction and detection were successfully fabricated using paper as a substrate. Viral RNA (e.g., zika, dengue, and chikungunya) was captured and eluted using a handheld RNA extraction paper-strip device, and another paper-chip device was used for reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay with a detection limit of a single copy and 10 copies of viral RNA in phosphate buffer solution (PBS) and serum, respectively. With these proposed devices, we have detected viral RNAs of zika and dengue in clinical human serum samples. The proposed paper-based extraction and detection platforms could be employed for detection of infectious viral diseases from complex clinical samples in resource-limited settings.

Integrated Bioaerosol Sampling/Monitoring Platform: Field-Deployable and Rapid Detection of Airborne Viruses.

Airborne pathogens causing infectious diseases are often highly transmittable between humans. Therefore, an airborne pathogen-monitoring system capable of on-site detection and identification would aid tremendously in preventing and controlling the early stages of pathogen spread. Here, we describe an integrated sampling/monitoring platform for on-site and real-time detection of airborne viruses. We used MS2 bacteriophage and avian influenza virus (AIV) H1N1 to evaluate bioaerosol sampling and detection performance of the platform. Our results show that, within 20 min, aerosolized viruses can be detected using the signal of near-infrared (NIR)-to-NIR nanoprobes. The pretreatment of the sampling pad improved the transfer efficiency of MS2 viruses to the detection zone, compared to an untreated pad. Our platform could detect concentrations as low as 104.294 50% egg infectious dose (EID50)/m3 AIVs collected from a cloacal swab sample (104.838 EID50/mL). These results indicate that our sampling/monitoring platform could be applied for the early detection of biological hazards in various fields.

A Paper-Based Device for Performing Loop-Mediated Isothermal Amplification with Real-Time Simultaneous Detection of Multiple DNA Targets.

Paper-based diagnostic devices have many advantages as a one of the multiple diagnostic test platforms for point-of-care (POC) testing because they have simplicity, portability, and cost-effectiveness. However, despite high sensitivity and specificity of nucleic acid testing (NAT), the development of NAT based on a paper platform has not progressed as much as the others because various specific conditions for nucleic acid amplification reactions such as pH, buffer components, and temperature, inhibitions from technical differences of paper-based device. Here, we propose a paper-based device for performing loop-mediated isothermal amplification (LAMP) with real-time simultaneous detection of multiple DNA targets. We determined the optimal chemical components to enable dry conditions for the LAMP reaction without lyophilization or other techniques. We also devised the simple paper device structure by sequentially stacking functional layers, and employed a newly discovered property of hydroxynaphthol blue fluorescence to analyze real-time LAMP signals in the paper device. This proposed platform allowed analysis of three different meningitis DNA samples in a single device with single-step operation. This LAMP-based multiple diagnostic device has potential for real-time analysis with quantitative detection of 102-105 copies of genomic DNA. Furthermore, we propose the transformation of DNA amplification devices to a simple and affordable paper system approach with great potential for realizing a paper-based NAT system for POC testing.

A structure-switchable aptasensor for aflatoxin B1 detection based on assembly of an aptamer/split DNAzyme.

An ultrasensitive, colorimetric and homogeneous strategy for aflatoxin B1 (AFB1) detection, which uses a DNA aptamer and two split DNAzyme halves, has been developed. Split halves of a hemin-binding DNAzymes is combined with an AFB1 aptamer to generate a homogeneous colorimetric sensor that undergoes an AFB1 induced DNA structural change. In the absence of AFB1, the split probes have peroxidase mimicking DNAzyme activity associated with catalysis of a color change reaction. Specific recognition of AFB1 by the aptamer component leads to structural deformation of the aptamer-DNAzyme complex, which causes splitting of the DNAzyme halves and a reduction in peroxidase mimicking activity. Therefore, a decrease of colorimetric signal arising from the catalytic process takes place upon in the presence of AFB1 in a concentration dependent manner in the 0.1-1.0 × 10(4) ng/mL range and with a colorimetric detection limit of 0.1 ng/mL. The new assay system exhibits high selectivity for AFB1 over other mycotoxins and can be employed detect the presence of AFB1 in ground corn samples. Overall, the strategy should serve as the basis for the development of rapid, simple and low-cost methods for detection of mycotoxins.