Surface-Initiated-Reversible-Addition–Fragmentation-Chain-Transfer Polymerization for Electrochemical DNA Biosensing

Abstract

Sensitive detection of biomolecules is integral for biomarker screening and early diagnosis. Herein, surface-initiated reversible-addition–fragmentation-chain-transfer (SI-RAFT) polymerization is exploited as a novel amplification strategy for highly sensitive electrochemical biosensing of DNA. Briefly, thiol-terminated peptide nucleic acid (PNA) probes are first self-assembled onto a gold electrode for the specific capture of target-DNA fragments; the carboxyl-group-containing dithiobenzoate 4-cyano-4-(phenylcarbonothioylthio)­pentanoic acid (CPAD) is then tethered to the hybridized PNA–DNA heteroduplexes by means of the well-established carboxylate–Zr4+–phosphate chemistry and serves as the chain-transfer agent (CTAs) for subsequent SI-RAFT polymerization, which is thermally initiated in the presence of 2,2′-azobis­[2-(2-imidazolin-2-yl)­propane] dihydrochloride (VA-044) as the free-radical initiator and ferrocenylmethyl methacrylate (FcMMA) as the monomer. Through SI-RAFT polymerization, one target-DNA fragment can be labeled by a large number of electroactive Fc tags, giving rise to significant amplification of the electrochemical signal. The SI-RAFT-polymerization-based strategy does not involve the use of natural enzymes or complex nanomaterials, offering the benefits of low cost and easy operation. Under optimal conditions, the electrochemical signal is linearly related to the logarithm of the concentration of target DNA over the range from 10 aM to 10 pM (R 2 = 0.997), with a detection limit down to 3.2 aM, which is much lower than those of other amplification-by-polymerization-based methods. By virtue of its easy operation, low cost, and high efficiency, the SI-RAFT-polymerization-based amplification strategy is believed to have great application prospects in the sensitive detection of biomolecules.

Radical polymerization reactions for amplified biodetection signals

“…In some cases, post-modification time is much longer than actual polymerization time as in AGET ATRP-based electrochemical biodetection, 34,35,38 where electroactive molecules are coupled to the generated polymer. Recent advances [41][42][43]63,64,68 demonstrate the use of electroactive monomers such as FMMA to reduce the amplification time.…”

“…In addition to enabling visual biodetection, thermally initiated RAFT polymerization has been proposed as an alternative signal amplification strategy to ATRP for electrochemical biosensors. 63,64 RAFT can reduce background signals from non-specific deposition of transition metals on electrodes, which may interfere with electrochemical measurements. Niu and coworkers demonstrated that thermally initiated RAFT polymerization could be exploited as a novel signal- amplifying tool for sensitive electrochemical detection of DNA (Fig.…”

“…6C). 63 Specifically, target DNA was captured by PNA on a gold electrode, and the PNA/DNA duplexes were treated with ZrOCl 2 and 4-cyano-4-( phenylcarbonothioylthio)pentanoic acid (CPAD) sequentially to attach the CTA by using phosphate-Zr 4+ -carboxylate chemistry. The CTA-functionalized substrate was soaked in a purged monomer solution containing ferrocenylmethyl methacrylate (FcMMA) and 2,2′-azobis[2-(2imidazolin-2-yl)propane] dihydrochloride (VA-044), which is selected as a thermal initiator because of its good water solubility and low decomposition temperature (44°C) for 10 h halflife.…”

In Situ Assembly of Nanomaterials and Molecules for the Signal Enhancement of Electrochemical Biosensors

“…However, the utilization of Cu 2+ ions as catalysts may interfere with the next electrochemical experiments because of the non-specific interaction with nucleic acids and the electrochemical deposition of metal on the electrode. For this consideration, they further explored novel surface-initiated electrochemically controlled reversible-addition-fragmentation-chain-transfer (SI-eRAFT) polymerization without the use of transition metal ions for assays [90][91][92]. For instance, they reported a signal-amplified electrochemical sensing of thrombin activity by SI-eRAFT polymerization [93].…”

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

“…26,27 Kong and co-workers have proposed various kinds of electrochemical sensor based on RAFT polymerization or ATRP polymerization for detection of sequence-specific DNAs and suggested that RAFT has better bio-compatibility as transition-metal, Cu 2+ of ATRP can cause toxicity. 28,29 Because RAFT polymerization is harmless to biomolecules 30 , it has great application prospects as a simple and effective signal amplification technique for biosensing for biomolecule detection. However, fluorescence detection DNA based on RAFT polymerization signal amplification has not been reported yet.…”