Boronate Affinity-Based Electrochemical Aptasensor for Point-of-Care Glycoprotein Detection

Abstract

As a class of oligosaccharide chain-containing proteins, glycoproteins are of great value in screening and early diagnosis of malignant tumors and other major diseases. Herein, we report a universal boronate affinity-based electrochemical aptasensor for point-of-care glycoprotein detection. Aptasensing of glycoproteins involves the specific recognition and capture of target glycoproteins by end-tethered nucleic acid aptamers and the site-specific labeling of ferrocene tags via the phenylboronic acid (PBA)-based boronate affinity interactions because the cis-diol sites of oligosaccharide chains on glycoproteins can selectively react with the PBA receptor groups to form cyclic phenylborates in aqueous basic media. Due to the presence of hundreds to thousands of cis-diol sites on a glycoprotein, a large number of ferrocene tags can be recruited for the signal-on aptasensing of glycoproteins at a low-abundance level, eliminating the need for extra amplification strategies. As a result, the boronate affinity-based electrochemical aptasensor is highly sensitive and selective for glycoprotein detection and tolerant to the false-positive results. The detection limit for α-fetoprotein (AFP) is 0.037 ng/mL, with a linear response ranging from 0.1 to 100 ng/mL. In addition to the merits of simple operation, short assay time, and low detection cost, the aptasensor is applicable to the detection of glycoproteins in serum samples and the point-of-care detection using disposable flexible electrodes. Overall, this work provides a universal and promising platform for the point-of-care detection of glycoproteins, holding great potential in screening and early diagnosis of glycoprotein-related malignant tumors and other major diseases.

Electrochemically Controlled Atom Transfer Radical Polymerization for Electrochemical Aptasensing of Tumor Biomarkers

“…The further increased steric hindrance caused by the capture of glycoprotein biomarkers (i.e., AFP) by aptamers led to the further decrease in redox currents (Figure A,d) and the further increase in R ct (Figure B,d). The lowest redox currents (Figure A,e) and the largest R ct (Figure B,e) were observed for the BrPBA/AFP/MCH/Apt/Au, indicative of the formation of the phenylborate anions . After eATRP, the redox currents increased significantly (Figure A,f) while the R ct decreased significantly (Figure B,f), indicating that the densely packed ferrocenyl polymers can greatly expedite the CT .…”

“…The attachment of ATRP initiators to the aptamer-captured targets was selectively mediated by the esterification crosslinking between the BA group and the cis -dihydroxyl sites of the conjugated oligosaccharide chains. In aqueous media with a pH value higher than the p K a of the BA group, the sp 2 -hybridized BA group can accept a hydroxyl ion to form the negatively charged sp 3 -hybridized −B­(OH) 3 − anion with which the reaction of the cis -dihydroxyl sites generates the stable cyclic phenylborates . In other words, the selective esterification crosslinking between the BA group and the cis -dihydroxyl sites is kinetically controlled by the environmental pH .…”

“…The linear regression equation can be expressed as y = 2.836lg x + 1.026 ( R 2 = 0.996) in which y is the current intensity in μA and x is the AFP concentration in pg/mL. The detection limit was 0.32 pg/mL, calculated according to 3σ blank /slope , in which σ blank is the standard deviation (SD) of the blank control. Relative to other methods as listed in Table S1, the detection limit of the eATRP-based electrochemical aptasensor is satisfactory.…”

“…The lowest redox currents (Figure 3A,e) and the largest R ct (Figure 3B,e) were observed for the BrPBA/AFP/MCH/ Apt/Au, indicative of the formation of the phenylborate anions. 56 After eATRP, the redox currents increased significantly (Figure 3A,f) while the R ct decreased significantly (Figure 3B,f), indicating that the densely packed ferrocenyl polymers can greatly expedite the CT. 55 The well-matched results of CV and EIS indicated that the electrode can be sequentially modified as expected.…”

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

“…As the phenylboronic acid (PBA) group can react with the 1,2- and 1,3- cis -diol sites of saccharides to form cyclic phenylborates, , PBA-containing materials have found much application in the separation and detection of saccharides and saccharide-containing substances. − For example, we have shown a boronate affinity-based electrochemical aptasensor for point-of-care (POC) detection of glycoproteins, taking advantage of the capture of target glycoproteins by nucleic acid aptamers and the decoration of glycoproteins with Fc signal tags via affinity coupling between the cis -diol site and the PBA group . As there are many cis -diol sites on a glycoprotein, many Fc signal tags can be recruited for amplified detection.…”