Label-free smartphone quantitation of bacteria by darkfield imaging of light scattering in fluoropolymer micro capillary film allows portable detection of bacteriophage lysis

Conventional methods for the detection and quantitation of bacteria are slow, laborious and require a laboratory. Microfluidic systems offer faster and portable testing, and smartphone cameras can record colorimetric or fluorometric bioassays, but this requires dye addition. Here, we demonstrate for the first time label-free smartphone detection of bacterial light scattering by darkfield microfluidic imaging to measure bacteria and bacteriophage lysis. A single LED and portable 3D printed imaging box allowed bacterial concentration and growth to be measured by direct imaging of bacterial light scattering. Bacteriophage lysis was detected within a 10-channel microfluidic device made from melt-extruded fluoropolymer micro capillary film, allowing rapid detection of host specificity. Elimination of unwanted reflections and optimising illumination angle are critical for successful darkfield bacterial imaging, with 15° giving maximal intensity. Bacterial sedimentation was directly observed within microfluidic devices, and detection sensitivity significantly increased by allowing bacteria to sediment for 30 min. With this simple, low-cost, 3D printed system bacterial concentrations down to an optical density of 0.1 could be measured corresponding to 8 × 104 colony forming units (CFU) per microdevice, approaching the sensitivity of conventional spectrophotometers. Bacteriophage lysis could be detected at a range of starting cell concentrations. With a low starting cell concentration, the increase in light scatter signal with incubation was prevented in the presence of bacteriophage. Conversely, with high starting cell concentration, the light scatter signal detected at the start was clearly eliminated when phage were added, indicating this simple system allows direct visualisation of bacteriophage eliminating light scattering by lysis

Streptococcus pneumoniae için DNA aptamerlerin geliştirilmesi ve karakterizasyonu

TEZ 616.014/DÖNsKaynakça: 92-100 ss.[Özet Yok

Selection of DNA aptamers to Streptococcus pneumonia and fabrication of graphene oxide based fluorescent assay

WOS:000441368000014PubMed:29964028Pneumococci are one of the leading causes of infections throughout the world causing problems mainly in children, elderly, and immune-deficient patients. In recent years antibiotic resistant Streptococcus pneumoniae strains become widespread. Therefore simple, rapid, and specific detection methods are needed for public health. In this study, DNA aptamer probes against S. pneumoniae were selected using bacterial Systematic Evolution of Ligands by Exponential Enrichment (SELEX) and these probes were integrated in to a graphene oxide (GO) based fluorescent assay. Among the tested aptamers three candidates Lyd-1, Lyd-2 and Lyd-3 showed K-d values of 844.7 +/- 123.6, 1984.8 +/- 347.5, and 661.8 +/- 111.3 nM, respectively. These candidates showed binding affinity to S. pneumoniae and no specific binding to the bacteria used in negative selection. The binding of aptamers were showed by fluorescence spectroscopy and flow cytometry. GO based label-free fluorescent assay developed using Lyd-3 aptamer had a unique detection limit of 15 cfu mL(-1) . Thus we believe that the selected aptamers and fabricated GO based assay has potential to be used in the detection of S. pneumoniae. Selected aptamers selectively bind to S. pneumonia with anti-pneumococcal potential and holds great potential to be used as molecular probes for identifying and targeting.Scientific and Technological Research Council of TurkeyTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [114S856]This work was supported by the Scientific and Technological Research Council of Turkey [Project No. 114S856]