A Fluorescent Sensor Based on Reversible Addition-Fragmentation Chain Transfer Polymerization for the Early Diagnosis of Non-small Cell Lung Cancer

Abstract

We proposed a novel, ultrasensitive and low-cost sensor using reversible addition-fragmentation chain transfer (RAFT) polymerization as a signal amplification strategy for the detection of CYFRA 21-1 DNA fragment, a tumor marker of non-small cell lung carcinoma. The peptide nucleic acid (PNA) probes were firstly immobilized on magnetic beads (MBs) to capture the CYFRA 21-1 DNA specifically. After hybridization, CPAD was tethered to the hetero duplexes through the carboxylate-Zr 4+ -phosphate chemistry.Subsequently, a number of fluorescent tags were introduced to the heteroduplexes through RAFT polymerization, leading to an amplification of the fluorescence signal. The sensor demonstrates a low limit of detection (LOD) of 0.02 fM. It has great selectivity with respect to base mismatch DNA, and high anti-interference ability in the normal human serum. Overall findings of the study suggest that proposed sensor holds enormous potential to be used as a tool for the early-stage diagnosis of the lung cancers. PNA (10 μL, 0.5 μM) was added and the reaction was performed at 37℃overnight for obtaining the PNA coated MBs (MBs/SSMCC/PNA). 31,32 The prepared MBs/SSMCC/PNA was incubated with the tDNA (20 μL, 0.1 nM) in PBST buffer (180 μL, pH 7.4) for 1.5 hours at 37 ℃. After washing with PBST buffer and magnetically separated, the MBs/SSMCC/PNA/tDNA was formed. Following the ZrOCl2 solution (20 μL, 5 mM, prepared with 15% EtOH) was added and the reaction was further conducted for 30 minutes at 37 ℃. Further, after washing, the MBs suspended in 180 μL PBST interacted with CPAD solution (20 μL, 0.5 mM) under similar conditions. MBs/SSMCC/PNA/tDNA/Zr 4+ /CPAD generated from the above reaction were washed with PBST buffer (0.1 M, pH 7.4) and magnetically separated. The FA solution (10 μL, 10 mM), VA-044 (20 μL, 40 μM), and 15% DMF (170 μL) were added respectively and the reaction was performed using a constant temperature shaker at 47 ℃ for 2 hours.The above-mentioned solution was magnetically separated, washed adequately, and finally resuspended in 3000 μL PBST buffer (0.1 M, pH 7.4) as a sample. The amplified fluorescent signal was acquired using Fluoro-spectrophotometer at an excitation wavelength of 489 nm, emission at 512 nm and a slit-width of 2 nm. Results and discussion Feasibility studyThe modified MBs were observed by confocal microscope. As shown in Fig. 1, No obvious fluorescence was observed on the PNA/Zr 4+ /CPAD/FA modified MBs and obvious Analytical SciencesAdvance Publication by J-STAGE

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