Real-time ligation chain reaction for DNA quantification and identification on the FO-SPR

Abstract

Different assays have been developed in the past years to meet point-of-care diagnostic tests requirements for fast and sensitive quantification and identification of targets. In this paper, we developed the ligation chain reaction (LCR) assay on the Fiber Optic Surface Plasmon Resonance (FO-SPR) platform, which enabled simultaneous quantification and cycle-to-cycle identification of DNA during amplification. The newly developed assay incorporated FO-SPR DNA melting assay, previously developed by our group. This required establishment of several assay parameters, including buffer ionic strength and thermal ramping speed as these parameters both influence the ligation enzyme performance and the hybridization yield of the gold nanoparticles (Au NPs) on the FO-SPR sensor. Quantification and identification of DNA targets was achieved over a wide concentration range with a calibration curve spanning 7 orders of magnitude and LOD of 13.75 fM. Moreover, the FO-SPR LCR assay could discriminate single nucleotide polymorphism (SNPs) without any post reaction analysis, featuring thus all the essential requirements of POC tests.

Fiber optic-SPR platform for fast and sensitive infliximab detection in serum of inflammatory bowel disease patients

“…Standard optical fibers have emerged over the years as excellent alternatives that can compete with the sensitivity and specificity of prism-based SPR sensors, while offering the advantage of low cost and small size greatly desired for developing a POC tool. One such platform, namely fiber optic surface plasmon resonance (FO-SPR), has been previously described by our group as a promising biosensor for protein-and DNA-based assays (Delport et al 2012;Janssen et al 2012;Knez et al 2013;Knez et al 2014;Pollet et al 2011;Pollet et al 2009;Tran et al 2011). The FO-SPR biosensor makes use of multimode optical fibers that facilitate SPR generation and enable implementation of bioassays.…”

Advances in ligase chain reaction and ligation-based amplifications for genotyping assays: Detection and applications

“…This assay has been implemented successfully for the detection of the oncogene Kras associated mutation. Two years later, Knez et al replaced the colorimetric detection method with fiber optic surface plasmon resonance (FO-SPR) platform to improve sensitivity with real-time monitoring of the assay in a simplified way without the need for any further analysis following the reaction [93]. Yin et al developed an ultrasensitive LCR based AuNPs biosensor for DNA Fig.…”

“…It is also inexpensive and stable with high signal/noise ratio. The whole assay takes around 4 h. AuNP enabled LCR is a portable, inexpensive technique that is extremely sensitive with real-time monitoring capability [42,93,94], It can detect as low as 1.5aM [94]. Preparation of probe coated gold nanoparticles takes 1 day and ligation assay takes less than an hour.…”

“…Preparation of probe coated gold nanoparticles takes 1 day and ligation assay takes less than an hour. Colorimetric methods, surface plasmon resonance or dynamic light scattering could be used for detection [42,[93][94]. The main drawbacks for this assay are lack of multiplexing potential and high-throughput applications.…”

Real-time fluorescence ligase chain reaction for sensitive detection of single nucleotide polymorphism based on fluorescence resonance energy transfer

“…However, these methods also require one or multiple steps after LCR amplification. Au nanoparticles and quantum dots have been developed as the indicators for LCR amplification (Yin et al, 2014;Chen et al, 2012;Knez et al, 2014;Zhu et al, 2014). Nevertheless, these methods need to label the LCR probes on the nanoparticles, in which the stability of the labels is challenging in many thermal cycles.…”