Cell‐free nucleic acids (cfNAs) are emerging diagnostic biomarkers for monitoring the treatment and recurrence of cancers. In particular, the biological role and clinical usefulness of cfNAs obtained from the plasma of patients with various cancers are popular and still intensely explored, yet most studies are limited by technical problems during cfNA isolation. A dimethyl dithiobispropionimidate (DTBP)‐based microchannel platform that enables spontaneous cfNA capture in 15 min with minimal cellular background and no requirements for use of bulky instruments is reported first. This platform identified KRAS and BRAF hot‐spot mutations following cfDNA isolation from the blood plasma and tissues obtained from 30 colorectal cancer patients. The correlation of mutations between the primary tissues and plasma from the patients was high using this platform with whole genome sequencing compared to the spin‐column method. This platform can also be combined with various detection approaches (biooptical sensor, Sanger sequencing, and polymerase chain reaction (PCR)) for rapid, simple, low‐cost, and sensitive circulating tumor DNA detection in blood plasma. The efficiency and versatility of this platform in isolating cfNAs from liquid biopsies has applications in cancer treatment and precision medicine.

Recently, RNA viral infections caused by respiratory viruses, such as influenza, parainfluenza, respiratory syncytial virus, coronavirus, and Middle East respiratory syndrome-coronavirus (MERS-CoV), and Zika virus, are a major public health threats in the world. Although myriads of diagnostic methods based on RNA amplification have been developed in the last decades, they continue to lack speed, sensitivity, and specificity for clinical use. A rapid and accurate diagnostic method is needed for appropriate control, including isolation and treatment of the patients. Here, we report an isothermal, label-free, one-step RNA amplification and detection system, termed as iROAD, for the diagnosis of respiratory diseases. It couples a one-step isothermal RNA amplification method and a bio-optical sensor for simultaneous viral RNA amplification/detection in a label-free and real-time manner. The iROAD assay offers a one-step viral RNA amplification/detection example to rapid analysis (<20min). The detection limit of iROAD assay was found to be 10-times more sensitive than that of real-time reverse transcription-PCR method. We confirmed the clinical utility of the iROAD assay by detecting viral RNAs obtained from 63 human respiratory samples. We envision that the iROAD assay will be useful and potentially adaptable for better diagnosis of emerging infectious diseases including respiratory diseases.

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Rapid and highly sensitive detection of biomolecules is greatly needed for pathogen diagnosis in clinical samples, but the method needs to be significantly improved in terms of sensitivity and specificity for actual use in clinical settings. Here, we report the development of an improved molecular diagnostics tool that utilizes CRISPR/dCas9mediated biosensor that couples a nuclease inactivated Cas9 (dCas9) and single microring resonator biosensor, enables label-free and real-time detection of pathogenic DNA and RNA. We addressed the clinical utility of this CRISPR/dCas9-mediated biosensor in tick-borne illnesses including scrub typhus (ST) and severe fever with thrombocytopenia syndrome (SFTS), whose clinical presentations are too similar to be easily differentiated. By using CRISPR/dCas9-mediated biosensor, we achieved single molecule sensitivity for the detection of ST (0.54 aM) and SFTS (0.63 aM); this detection sensitivity is 100 times more sensitive than that of RT-PCR assay. Finally, CRISPR/dCas9-mediated biosensor was able to clearly distinguish between ST and SFTS in serum samples within 20 min. We believe that CRISPR/dCas9-mediated biosensor will be useful for rapid and accurate molecular diagnostic tool that is suitable for immediate clinical applications.

Diseases caused by pathogenic microorganisms including bacteria and viruses can cause serious medical issues including death and result in huge economic losses. Despite the myriad of recent advances in the rapid and accurate detection of pathogens, large volume clinical samples with a low concentration of pathogens continue to present challenges for diagnosis and surveillance. We here report a simple and label-free approach via homobifunctional imidoesters (HIs) with a microfluidic platform (SLIM) to efficiently enrich and extract pathogens at low concentrations from clinical samples. The SLIM system consists of an assembled double microfluidic chip for streamlining large volume processing and HIs for capturing pathogens and isolating nucleic acids by both electrostatic and covalent interaction without a chaotropic detergent or bulky instruments. The SLIM system significantly increases the enrichment and extraction rate of pathogens (up to 80% at 10 CFU (colony forming unit) in a 1 mL volume within 50 min). We demonstrated its clinical utility in large sample volumes from 46 clinical specimens including environmental swabs, saliva, and blood plasma. The SLIM system showed higher sensitivity with these samples and could detect pathogens that were below the threshold of detection with other methods. Finally, by combining our SLIM approach with an isothermal optical sensor, pathogens could be detected at a very high sensitivity in blood plasma samples within 80 min via enrichment, extraction and detection steps. Our SLIM system thus provides a simple, reliable, cost-effective and ultrasensitive pathogen diagnosis platform for use with large volume clinical samples and would thus have significant utility for various infectious diseases.

Versatile, simple and efficient sample preparation is desirable for point-of-care testing of emerging diseases such as zoonoses, but current sample preparation assays are insensitive, labour-intensive and time-consuming and require multiple instruments. We developed a single-tube sample preparation approach involving direct pathogen enrichment and extraction from human specimens using diatomaceous earth (DE). Amine-modified DE was used to directly enrich a zoonotic pathogen, Brucella, in a large sample volume. Next, a complex of amine-modified DE and dimethyl suberimidate was used for nucleic acid extraction from the enriched pathogen. Using our single-tube approach, the pathogen can be enriched and extracted within 60min at a level of 1 colony formation unit (CFU) from a 1ml sample volume in the same tube. The performance of this approach is 10-100 times better than that of a commercial kit (10 to 10CFU/ml) but does not require a large centrifuge. Finally, we combined the single-tube approach with a bio-optical sensor for rapid and accurate zoonotic pathogen detection in human urine samples. Using the combination system, Brucella in human urine can be efficiently enriched (~ 8-fold) and the detection limit is enhanced by up to 100 times (1CFU/ml bacteria in urine) compared with the commercial kit. This combined system is fast and highly sensitive and thus represents a promising approach for disease diagnosis in the clinical setting.

Rapid identification of emerging infectious pathogens is crucial for preventing public health threats. Various pathogen detection techniques have been introduced; however, most techniques are time-consuming and lack multiple-target detection specificity. Although multiple-target detection techniques can distinguish emerging infectious pathogens from related pathogens, direct amplification methods have not been widely examined. Here, we present a novel arch-shaped multiple-target sensor capable of rapid pathogen identification using direct amplification in clinical samples. In this study, an arch-shaped amplification containing primer sequences was designed to rapidly amplify multiple targets. Further, the sensing platform allowed for sensitive and specific detection of human coronavirus, Middle East respiratory syndrome, Zika virus, and Ebola virus down to several copies. This platform also simultaneously distinguished between Middle East respiratory syndrome and human coronavirus in clinical specimens within 20 min. This arch-shaped multiple-target sensing assay can provide rapid, sensitive, and accurate diagnoses of emerging infectious diseases in clinical applications.

iNAD is a valuable, rapid method of detecting SFTS virus and with high clinical sensitivity and specificity.