Thin-film technology for direct visual detection of nucleic acid sequences: applications in clinical research

Abstract

Certain optical conditions permit the unaided eye to detect thickness changes on surfaces on the order of 20 A, which are of similar dimensions to monomolecular interactions between proteins or hybridization of complementary nucleic acid sequences. Such detection exploits specific interference of reflected white light, wherein thickness changes are perceived as surface color changes. This technology, termed thin-film detection, allows for the visualization of subattomole amounts of nucleic acid targets, even in complex clinical samples. Thin-film technology has been applied to a broad range of clinically relevant indications, including the detection of pathogenic bacterial and viral nucleic acid sequences and the discrimination of sequence variations in human genes causally related to susceptibility or severity of disease.

Deoxyribozyme Cascade for Visual Detection of Bacterial RNA

“…[1] The concentration of total RNA was calculated by measuring its absorption at 260 nm using a Nanodrop spectrophotometer and taking into account that 1 OD 260 corresponds to 40 μg/mL. The stock concentrations were 660 ng/μL and 1400 ng/μl for M. bovis and M. smegmatis RNA, respectively.…”

“…It should report the presence of a specific analyte with high accuracy in a simple and straightforward assay. [1] An example of a near-ideal sensor is the home pregnancy test, in which the exposure of an immunochromatographic strip to a urine sample containing a pregnancy marker, human chorionic gonadotropin, develops a colored test line. The important characteristics of the sensor include visual signal output that can be detected by the naked eye, and a simple protocol that does not require user expertise.…”

“…This LOD is at least 20 times lower than that reported for PDz-based sensor without the Dz cascade. [1d,3a-d] Since an instrument-free detection format is preferable for the POC diagnostics, we explored TLC plate-based signal visualization. For this purpose, 3 μL of the final samples were spotted on a TLC plate.…”

“…Binary deoxyribozyme probes were found to have exceptional discrimination ability toward nucleotide substitutions in nucleic acids. [1d,2] This is due to the possibility to design one of the two analyte-binding arms to be selective. In the present design, the analyte-binding arm of Dz b strand was chosen to target a hyper-variable fragment of 16S rRNA (Figure 2A).…”

Staph ID/R: a Rapid Method for Determining Staphylococcus Species Identity and Detecting the mecA Gene Directly from Positive Blood Culture

“…The test utilizes multiplex, isothermal helicase-dependent amplification (HDA) of target DNA sequences (1) in the mecA gene, which encodes methicillin resistance, and in the species-variable tuf gene, which encodes the polypeptide elongation factor Tu in bacteria (25). Amplified products are detected by species-specific probes arrayed on a modified silicon chip such that intermolecular interactions trigger colorimetric intensity changes that permit visual detection of attomole quantities of nucleic acids (14). We performed analytical characterization of the test using seeded blood cultures with known bacterial species and validated its performance by testing 104 positive clinical blood cultures determined to contain Grampositive cocci in clusters.…”