RESUMO
We report on the first efforts to isolate acidic RNA-cleaving DNAzymes (aRCDs) from a random-sequence DNA pool by in vitro selection that are activated by a microbe Escherichia coli (E. coli), at pH 5.3. Importantly, these E. coli-responsive aRCDs only require monovalent metal ions as cofactors for cleaving a fluorogenic chimeric DNA/RNA substrate. Such characteristics can be used to efficiently protect RCDs from both intrinsic chemical instability and external enzymatic degradation. One remarkable DNAzyme, aRCD-EC1, is specific for E. coli, and its target is likely a protein. Furthermore, truncated aRCD-EC1 had significantly improved catalytic activity with an observed rate constant (kobs) of 1.18 min-1, making it the fastest bacteria-responding RCD reported to date. Clinical evaluation of this aRCD-based fluorescent assay using 40 patient urine samples demonstrated a diagnostic sensitivity of 100% and a specificity of 100% at a total analysis time of 50 min without a bacterial culture. This work can expand the repertoire of DNAzymes that are active under nonphysiological conditions, thus facilitating the development of diverse DNAzyme-based biosensors in clinical diagnosis.
Assuntos
Técnicas Biossensoriais , DNA Catalítico , Humanos , DNA Catalítico/química , Escherichia coli/metabolismo , DNA/química , RNA/química , MetaisRESUMO
We reported a colorimetric paper-based device by integrating the modified acid RNA-cleaving DNAzymes (MaRCD-EC1) for highly sensitive (detection limit = 102 CFU mL-1), and rapid (within 30 min) detection of E. coli without amplification. This device exhibited a clinical sensitivity of 100% and a specificity of 100% in identifying E. coli-associated urinary tract infections (UTIs) using the clinical urine samples.
Assuntos
Colorimetria , DNA Catalítico , Escherichia coli , Papel , DNA Catalítico/química , DNA Catalítico/metabolismo , Escherichia coli/isolamento & purificação , Infecções Urinárias/diagnóstico , Infecções Urinárias/microbiologia , Infecções Urinárias/urina , Humanos , Limite de Detecção , Técnicas Biossensoriais/métodos , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/urinaRESUMO
A novel and simple technique for the measurement of time delay in the optical fiber by a free-running laser is proposed and demonstrated. The fiber to be measured was spliced to an erbium-doped fiber so as to form a ring-cavity laser. The mode beating frequency of the laser was measured to determine the round-trip time delay. This precise and efficient technique has an accuracy of 10(-8) for a fiber length of 100 km and of 10(-6) for a several-meters-long one.