Abstract
Gene regulation systems are mimicked by simple quantitative detection of non-nucleic acid molecular targets such as protein and metabolite. Here, we describe a one-tube, one-step real-time quantitative detection methodology for isothermal signal amplification of those targets. Using this system, real-time quantitative detection of thrombin and streptomycin, which were used as examples for protein and metabolite targets, was successfully demonstrated with detection limits of at most 50 pM and 75 nM, respectively. Notably, the dynamic range of target concentrations could be obtained for over four orders of magnitude. Thus, our method is expected to serve as a point-of-care or on-site test for medical diagnosis and food and environmental hygiene.
Highlights
To this end, we employed split aptamers[14,15,16,17,18] for target recognition and modified a φ29 DNA polymerase-catalyzed rolling circle amplification (RCA) system[19] termed “signal amplification by ternary initiation complexes (SATIC)”[20] as a platform of amplicon production
RCA in our light-up system only worked at low concentrations up to 7.5 mM and 10 mM of Na+ and K+, respectively (Figure S2)
RCA running and aptamer activities could simultaneously be sustained under the optimal condition of 10 mM of K+ (Figures S3 and S4)
Summary
Hiroto Fujita[1], Yuka Kataoka[1], Remi Nagano[1], Yasuyo Nakajima[2], Masanobu Yamada[2], Naoki Sugimoto3,4 & Masayasu Kuwahara 1. We envisioned that, as with the real-time immuno-polymerase chain reaction (RT-IPCR) method[12,13], the presence of the target molecule can be converted into the production of amplified polynucleotide strands, which can quantitatively be detected as a fluorescent signal; the detection reaction isothermally proceeds in a one-tube, one-step manner, i.e., without the requirement of temperature fluctuations or washing steps To this end, we employed split aptamers[14,15,16,17,18] for target recognition and modified a φ29 DNA polymerase-catalyzed rolling circle amplification (RCA) system[19] termed “signal amplification by ternary initiation complexes (SATIC)”[20] as a platform of amplicon production. In principle, if the target is present, the test tubes are expected to emit fluorescence after adding the relevant reagents after maintenance of an isothermal temperature (37 °C)
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