These

pitfalls include high cost, requirement of expert personnel, advanced instruments, and much time [20]. Thus, in addition to qualitative iPCR and other similar methods, iLAMP-Au-nanoprobe method can be used instead of real-time iPCR. Different nanoparticle-based nanoprobes have been designed so far. Gold, silver, and HCS assay quantum dot (fluorescent) nanoparticles are main nanoparticles that are used for detection of target nucleic acids. Gold nanoprobes (Au nanoprobes) Gold-nanoparticle probes take the optical advantages of gold nanoparticles at the time of specific hybridization between their nucleic acid parts with target nucleic acids. The hybridization brings the gold nanoparticle part of these probes near each other, leading their aggregation and subsequent color change from deep red to blue/purple [38]. Generally, two main formats are used to detect target DNA by Au nanoprobes called ‘homogenous’ or ‘solution-based’ format and ‘heterogenous’ or ‘solid-based’ format. In the homogenous format, the hybridization of Au nanoprobes with target occurs homogenously without any attachment to any solid supports. However, in the heterogenous format the recognition

of target sequence occurs on a specific probe sequence linked to a solid support. The routine method of target detection using heterogenous format is a sandwich-type reaction, in which target sequence is hybridized with two specific probes, so that one probe is attached to a solid base; after hybridization of target sequence,

Janus kinase (JAK) check details the secondary probe (Au nanoprobe) hybridizes with the other part of target sequence. The presence of specific target is detected then by the use of silver enhancement. This format of detection shows more sensitivity and specificity for recognition of target nucleic acid compared with homogenous format [38]. Although silver enhancement has some drawbacks, the results can be quantified based on the intensities of reduced silver. The drawbacks are high background of silver enhancement and weak signal-to-noise ratio [39]. However, these can be avoided by using gold enhancement instead of silver enhancement. Gold enhancement has been used in two studies for detecting target nucleic acid in homogenous format [40, 41]; and due to the high false-positive results associated with silver enhancement [39], gold enhancement can be used for the detection of target nucleic acids in heterogenous format and can be applied for quantitative detection of iLAMP products by Au nanoprobes in iLAMP-Au-nanoprobe method. Another advantage of heterogenous format is its applicability for simultaneous, high-throughput assay of several samples. This can be achieved using 96-well or 384-well microplates so that each single well can be a site for one reaction. Another type of solid-base format is the application of paper strips for detection of targets by Au nanoprobes [34].