A label-free, colorimetric method has been developed for ultrasensitive detection of nucleic acids that is based on photoinduced silver ion (Ag(+)) reduction around DNA bases. The assay system is capable of directly detecting bacterial genomic DNA without the need for PCR amplification.
We developed a whole-cell surface plasmon resonance (SPR) sensor based on a leucine auxotroph of Escherichia coli displaying a gold-binding protein (GBP) in response to cell growth and applied this sensor to the diagnosis of maple syrup urine disease, which is represented by the elevated leucine level in blood. The leucine auxotroph was genetically engineered to grow displaying GBP in a proportion to the concentration of target amino acid leucine. The GBP expressed on the surface of the auxotrophs directly bound to the golden surface of an SPR chip without the need for any additional treatment or reagents, which consequently produced SPR signals used to determine leucine levels in a test sample. Gold nanoparticles (GNPs) were further applied to the SPR system, which significantly enhanced the signal intensity up to 10-fold by specifically binding to GBP expressed on the cell surface. Finally, the diagnostic utility of our system was demonstrated by its employment in reliably determining different statuses of maple syrup urine disease based on a known cutoff level of leucine. This new approach based on an amino acid-auxotrophic E. coli strain expressing a GBP that binds to an SPR sensor holds great promise for detection of other metabolic diseases of newborn babies including homocystinuria and phenylketonuria, which are also associated with abnormal levels of amino acids.
A simple, colorimetric method is developed for the determination of deoxyribonuclease I (DNase I) activity based on the novel finding that DNase I can promote the photoinduced synthesis of gold nanoparticles (AuNPs). In the absence of DNase I, a phosphorothioate (PS) DNA probe remains intact and captures Au(iii) through a strong Au-thiol interaction, which prevents the photoinduced synthesis of AuNPs, leaving the sample in a colorless state. On the other hand, in the presence of DNase I, the PS DNA probe is cleaved into small fragments that are removed via a simple purification process. The resulting solution, after the incubation with HAuCl and threonine (Thr), forms AuNPs by UV light irradiation with the aid of Thr which acts as a catalyst for the Au(iii) reduction process. As a result, a red-colored suspension is produced. By monitoring the color changes of the samples with the naked eye, the DNase I activity was conveniently determined. In addition, the clinical utility of this simple, yet highly efficient colorimetric strategy was verified by reliably quantifying the DNase I activities in a bovine urine sample. Importantly, the working principle designed for the determination of DNase I activity was successfully expanded for the detection of target nucleic acids, ensuring the universal applicability of the developed assay system.
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