Simple and label-free pathogen enrichment via homobifunctional imidoesters using a microfluidic (SLIM) system for ultrasensitive pathogen detection in various clinical specimens

Abstract

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.

A Simple Microfluidic Assay for Diagnosing Tuberculous Meningitis in HIV-Uninfected Patients

“…In addition, our system does not require additional instruments or specific antibodies or probes for sample processing. Furthermore, the assembled microfluidic chips that we used to streamline sample processing are potentially scalable at low cost, and this would increase the surface area for pathogen capture without increasing the size of the device (8). Thus, we can perform this test in a simple and efficient way, and we expect that it will be usable in resource-limited countries.…”

“…However, these 5 patients did not have histories of previous TB. In addition, because the SLIM assay uses a single platform for enrichment and extraction of pathogens, the risk of external contamination should be reduced (8). Thus, we think that the likelihood of false positives is extremely low.…”

“…The SLIM assay. The principle of the SLIM assay, and its components, have been described previously (8). Briefly, it is based on combining a microfluidic platform with a low-cost thin film and homobifunctional imidoester (HI) reagents to enrich and extract M. tuberculosis.…”

“…Hence, it is important to develop a new diagnostic tool to rapidly rule out TBM. We have developed a new assay for diagnosing TBM, involving simple and label-free pathogen enrichment by homobifunctional imidoesters (HIs) using a microfluidic system, called the SLIM assay (8). We have evaluated the diagnostic performance of the SLIM assay followed by real-time PCR in HIV-uninfected patients with suspected TBM compared with the uniform case definition (9) in a country with an intermediate tuberculosis (TB) burden and low HIV burden.…”

An ultraportable and versatile point-of-care DNA testing platform

“…Versatility is another great challenge for POCT. The ideal DNA POCT system needs not only to be able to process diverse types of samples (such as saliva, urine, and blood) but also to be able to detect the multiple forms of DNA targets including pathogen-specific DNA sequences, gene mutations, single-nucleotide polymorphisms (SNPs), and allele genes (13)(14). So far, it is still difficult to detect pathogenspecific DNA directly from raw samples (e.g., saliva) in the fashion of "sample-in-result-out" (15).…”