Boronate-Affinity Cross-Linking-Based Ratiometric Electrochemical Detection of Glycoconjugates

Abstract

Despite the widespread application of the boronate-affinity cross-linking (BAC) in the separation, enrichment, and sensing of glycoconjugates, it remains a huge challenge to integrate the BAC into the selective electrochemical detection of glycoconjugates due to the poor selectivity of the BAC. Herein, we demonstrate a BAC-based ratiometric electrochemical method for the simple, low-cost, and highly sensitive and selective detection of glycoconjugates. Briefly, the methylene blue (MB)-tagged nucleic acid aptamer is exploited as the recognition element to selectively capture target glycoconjugate, to which a large number of ferrocene (Fc) tags are subsequently labeled via the BAC between the phenylboronic acid (PBA) group and the cis-diol site of the oligosaccharide chains on the captured targets. Using the MB tag as the internal reference and the Fc tag as the reporter of the target capture, the dual-signal output enables the ratiometric detection. Due to the presence of a high density of the cis-diol sites on a glycoconjugate, sufficiently high sensitivity can be obtained even without using any amplification strategies. Using glycoprotein mucin 1 (MUC1) as the model target, the signal ratio (I Fc/I MB) exhibits good linearity over the range from 0.05 to 50 U/mL, with a detection limit of 0.021 U/mL. In addition to the high sensitivity and selectivity, the results of the analysis of MUC1 in serum samples are acceptable. By virtue of its simplicity, cost-effectiveness, and high robustness and reproducibility, this BAC-based ratiometric electrochemical method holds great promise in the highly sensitive and selective detection of glycoconjugates.

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

“…As seen, the cathodic peak current exhibited satisfactory linearity when the target concentration was varied from 10 fM to 10 nM, with a regression equation of y (μA) = −0.490lg­( x /pM) – 1.857 ( R 2 = 0.996). The detection limit of the polysaccharide-amplified method was calculated to be about 2.9 fM (3σ/slope, ,, n = 5), which is satisfactory when compared with other methods involving signal amplification by enzymes, polymers, or nanomaterials, as listed in Table S2. The high detection sensitivity was imparted by the polysaccharide-based signal amplification because each polysaccharide chain can be decorated with many Fc signal tags.…”

“…Obviously, the optimal FcPBA concentration was 0.25 mM. The lower cathodic peak current observed at a higher FcPBA concentration is likely due to the formation of the hydrogen bond-linked PBA networks. , …”

“…On the contrary, the pH above the p K a of the PBA group would result in the formation of the sp 3 -hybridized PhB­(OH) 3 – anion . Previous reports have shown that the anionic form can react with cis -diols at a much higher rate relative to the neutral form, while the stability of the tetrahedral phenylboronate anions is much higher than that of the neutral trigonal planar phenylboronate esters. ,, That is, the affinity coupling between the 1,2- and 1,3- cis -diols and the PBA group is highly pH-dependent. − In view of this, the effect of pH on the cathodic peak current was studied (Figure S2B). As is shown, the optimal pH for the affinity coupling was 8.0, which was therefore adopted in the subsequent experiments.…”

“…For the Zr/DNA/MCH/PNA/Au and PGA/Zr/DNA/MCH/PNA/Au, the increased R ct and the decreased redox currents can be ascribed to the steric hindrance. As a result of the electrostatic repulsion from the phenylborate anions to the [Fe­(CN) 6 ] 3–/4– couples, , the decoration of polysaccharide chains with FcPBAs resulted in the further increase of R ct as well as the further decrease of the redox currents. According to the above results, it can be concluded that the FcPBA/PGA/Zr/DNA/MCH/PNA/Au was fabricated as expected.…”

“…33 Previous reports have shown that the anionic form can react with cis-diols at a much higher rate relative to the neutral form, while the stability of the tetrahedral phenylboronate anions is much higher than that of the neutral trigonal planar phenylboronate esters. 33,38,46 That is, the affinity coupling between the 1,2-and 1,3-cis-diols and the PBA group is highly pH-dependent. 46−48 In view of this, the effect of pH on the cathodic peak current was studied (Figure S2B).…”