Electrochemical Detection of Femtomolar DNA via Boronate Affinity-Mediated Decoration of Polysaccharides with Electroactive Tags

Abstract

In view of their high efficiency and cost-effectiveness, polymers are of great promise as carriers for signal tags in amplified detection. Herein, we present a polysaccharide-amplified method for the electrochemical detection of a BRCA1 breast cancer gene-derived DNA target at the femtomolar levels. Briefly, peptide nucleic acid (PNA) with a complementary sequence was tethered as the capture probe for the DNA target, to which carboxyl groupcontaining polysaccharides were then attached via facile phosphate-Zr(IV)-carboxylate crosslinking, followed by the decoration of polysaccharide chains with electroactive ferrocene (Fc) signal tags via affinity coupling between a cis-diol site and phenylboronic acid (PBA) group. As the polysaccharide chain contains hundreds of cisdiol sites, boronate affinity can enable the site-specific decoration of each polysaccharide chain with hundreds of Fc signal tags, efficiently transducing each target capture event into the decoration of many Fc signal tags. As polysaccharides are cheap, renewable, ubiquitous, and biodegradable natural biopolymers, the use of polysaccharides for signal amplification offers the benefits of high efficiency, cost-effectiveness, excellent biocompatibility, and environmental friendliness. The linear range of the polysaccharideamplified method for DNA detection was demonstrated to be from 10 fM to 10 nM (R 2 = 0.996), with the detection limit as low as 2.9 fM. The results show that this method can also discriminate single base mismatch with satisfactory selectivity and can be applied to DNA detection in serum samples. In view of these merits, the polysaccharide-amplified PNA-based electrochemical method holds great promise in DNA detection with satisfactory sensitivity and selectivity.

Boronate Affinity-Amplified Electrochemical Aptasensing of Lipopolysaccharide

“…As the cathodic peak current increased with PBA-Fc concentration from 0.05 to 0.25 mM and then decreased with the further increase of PBA-Fc concentration (Figure S3B), the optimal PBA-Fc concentration was 0.25 mM. As the PBA group can form the intermolecular hydrogen bond, , we reasoned that the decrease in the cathodic peak current at higher PBA-Fc concentration (>0.25 mM) might be due to the formation of the hydrogen bond-crosslinked PBA-Fc aggregates . In the following experiments, the concentration of 0.25 mM was applied for the decoration of LPS with PBA-Fc.…”

“…For the GE/Apt/MCH/LPS/PBA-Fc, a high cathodic peak current can be observed at 0.43 V. For the GE/Apt/MCH/LPS, on the contrary, no cathodic peak was observed. Obviously, the cathodic peak current was derived from the Fc signal tags, corresponding to the 1e reduction of Fc + into Fc . Also, almost no cathodic peak can be observed for the GEs without modifying the LPS aptamer or LPS.…”

“…The BA-mediated crosslinking is a pH-dependent process . In aqueous solutions, with the pH above the corresponding p K a , the PBA group is present in the form of the sp 3 -hybridized tetrahedral anion (i.e., PhB­(OH) 3 – ), which can react efficiently with cis -diols to form the stable phenylboronate anions . Instead, the pH lower than the p K a of the PBA group results in the formation of the neutral, sp 2 -hybridized trigonal structure (i.e., PhB­(OH) 2 ), which cannot form stable complexes with cis -diols, while the crosslinking kinetics is significantly lower. , Considering this, the effect of pH for the BA-mediated crosslinking on the cathodic peak current was investigated.…”

“…Inspired by this, we carefully investigated the structure of the polysaccharide chain of LPS and found that most of the monosaccharide residues of LPS’s polysaccharide chain contain at least a cis -diol site . As the cis -diols can crosslink selectively with the boronic acid group to form boronates in aqueous solutions, − the boronate affinity (BA)-mediated crosslinking can be harnessed for the direct decoration of polysaccharide chains with signal tags. , Very recently, we have illustrated a polysaccharide-amplified method for the electrochemical detection of femtomolar DNA via the BA-mediated decoration of polysaccharide chains with ferrocene signal tags . Specifically, the polysaccharide chains were preattached to the DNA targets that were captured by the peptide nucleic acid (PNA) probes and then the BA-mediated crosslinking was utilized for the site-specific decoration of polysaccharide chains with a large number of signal tags.…”

“…42,43 Very recently, we have illustrated a polysaccharideamplified method for the electrochemical detection of femtomolar DNA via the BA-mediated decoration of polysaccharide chains with ferrocene signal tags. 44 Specifically, the polysaccharide chains were preattached to the DNA targets that were captured by the peptide nucleic acid (PNA) probes and then the BA-mediated crosslinking was utilized for the sitespecific decoration of polysaccharide chains with a large number of signal tags. As the decoration of polysaccharide chains with ferrocene signal tags can be fulfilled within 15 min in aqueous solutions, the BA-mediated crosslinking is highly efficient and biologically friendly.…”