Colorimetric Detection of DNA Strands on Cellulose Microparticles Using ZZ-CBM Fusions and Gold Nanoparticles.

Nucleic acid testing requires skilled personnel and expensive instrumentation. A method for the colorimetric detection of oligonucleotides that combines cellulose microparticles with biomolecular recognition is presented. DNA sequences from Trypanosoma brucei and dengue are used as model targets. Cellulose microparticles (≈20 µm) are bioactived by anchoring anti-biotin antibodies via fusions that combine a carbohydrate-binding module (CBM) with the ZZ fragment of protein A. Samples are prepared by incubating DNA probes immobilized on ≈14 nm gold nanoparticles (AuNPs) with biotin-labeled targets and mixed with bioactive microparticles. The presence of unlabeled targets could also be probed by introducing a second, biotinylated DNA probe. The target:probe-AuNP hybrids are mixed with and captured by the microparticles, which change color from white to red. Depletion of AuNPs from the liquid is also signaled by a decrease in absorbance at 525 nm. It was possible to detect targets with concentrations as low as 50 n m. In the presence of noncomplementary targets, microparticles remain white and the liquid remains red. The system is able to discriminate targets with a high degree of homology (≈53%). Overall, it is demonstrated that simple systems for the visual detection of nucleic acids can be set up by combining cellulose microparticles with biomolecular recognition agents based on CBMs and AuNPs.

Capture and detection of DNA hybrids on paper via the anchoring of antibodies with fusions of carbohydrate binding modules and ZZ-domains.

Microfluidic paper-based analytical devices (µPADs) fabricated by wax-printing are suitable platforms for the development of simple and affordable molecular diagnostic assays for infectious diseases, especially in resource-limited settings. Paper devices can be modified for biological assays by adding appropriate reagents to the test areas. For this purpose, the use of affinity immobilization strategies can be a good solution for bioactive paper fabrication. This paper describes a methodology to capture labeled-DNA strands and hybrids on paper via the anchoring of antibodies with a fusion protein that combines a family 3 carbohydrate binding module (CBM) from Clostridium thermocellum, with high affinity to cellulose, and the ZZ fragment of the staphyloccocal protein A, which recognizes IgG antibodies via their Fc portion. Antibodies immobilized via CBM-ZZ were able to capture appropriately labeled (biotin, fluorescein) DNA strands and DNA hybrids. The ability of an antibody specific to biotin to discriminate complementary from noncomplementary, biotin-labeled targets was demonstrated in both spot and microchannel assays. Hybridization was detected by fluorescence emission of the fluorescein-labeled DNA probe. The efficiency of the capture of labeled-DNA by antibodies immobilized on paper via the CBM-ZZ construct was significantly higher when compared with a physical adsorption method where antibodies were simply spotted on paper without the intermediation of other molecules. The experimental proof of concept of wax-printed µPADs functionalized with CBM-ZZ for DNA detection at room temperature presented in this study constitutes an important step toward the development of easy to use and affordable molecular diagnostic tests.