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Amplified voltammetric detection of glycoproteins using 4-mercaptophenylboronic acid/biotin-modified multifunctional gold nanoparticles as labels.

Liu L, Xing Y, Zhang H, Liu R, Liu H, Xia N - Int J Nanomedicine (2014)

Bottom Line: The MBA-biotin-AuNPs facilitated the attachment of streptavidin-conjugated alkaline phosphatase for the production of electroactive p-aminophenol from p-aminophenyl phosphate substrate.A detection limit of 8 fmol L(-1) for rHuEPO detection was achieved.Other glycosylated and non-glycosylated proteins, such as horseradish peroxidase, prostate specific antigen, metallothionein, streptavidin, and thrombin showed no interference in the detection assay.

View Article: PubMed Central - PubMed

Affiliation: College of Chemistry and Chemical Engineering, Anyang Normal University, Anyang, Henan, People's Republic of China ; College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan, People's Republic of China.

ABSTRACT
Ultrasensitive detection of protein biomarkers is essential for early diagnosis and therapy of many diseases. Glycoproteins, differing from other types of proteins, contain carbohydrate moieties in the oligosaccharide chains. Boronic acid can form boronate ester covalent bonds with diol-containing species. Herein, we present a sensitive and cost-effective electrochemical method for glycoprotein detection using 4-mercaptophenylboronic acid (MBA)/biotin-modified gold nanoparticles (AuNPs) (MBA-biotin-AuNPs) as labels. To demonstrate the feasibility and sensitivity of this method, recombinant human erythropoietin (rHuEPO) was tested as a model analyte. Specifically, rHuEPO was captured by the anti-rHuEPO aptamer-covered electrode and then derivatized with MBA-biotin-AuNPs through the boronic acid-carbohydrate interaction. The MBA-biotin-AuNPs facilitated the attachment of streptavidin-conjugated alkaline phosphatase for the production of electroactive p-aminophenol from p-aminophenyl phosphate substrate. A detection limit of 8 fmol L(-1) for rHuEPO detection was achieved. Other glycosylated and non-glycosylated proteins, such as horseradish peroxidase, prostate specific antigen, metallothionein, streptavidin, and thrombin showed no interference in the detection assay.

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(A) Differential pulse voltammograms for the detection of different concentrations of rHuEPO. (B) Plots of the Ipa against the rHuEPO concentration (0.02–10 pmol L−1). The inset shows the linear plots at concentrations of 0.02, 0.2, 0.5, 1.0, and 2.0 pmol L−1.Note: Each point was averaged from three replicates, and the error bars show the absolute standard.Abbreviation: rHuEPO, recombinant human erythropoietin.
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f4-ijn-9-2619: (A) Differential pulse voltammograms for the detection of different concentrations of rHuEPO. (B) Plots of the Ipa against the rHuEPO concentration (0.02–10 pmol L−1). The inset shows the linear plots at concentrations of 0.02, 0.2, 0.5, 1.0, and 2.0 pmol L−1.Note: Each point was averaged from three replicates, and the error bars show the absolute standard.Abbreviation: rHuEPO, recombinant human erythropoietin.

Mentions: Differential pulse voltammetry is more sensitive than cyclic voltammetry, since it can decrease the background charging currents. Furthermore, we assessed the linear range, detection limit, and selectivity of this method with differential pulse voltammetry. The differential pulse voltammograms obtained at different rHuEPO concentrations are shown in Figure 4A. As a result, we found that the current increased linearly with the rHuEPO concentration in the range of 0.02–2.0 pmol L−1 and began to level off beyond 2 pmol L−1 (Figure 4B). All the relative standard deviations were found to be below 9.5%. The linear regression equation was expressed as Ipa (μA) =0.038+0.14 [rHuEPO] (pmol L−1) (R2=0.99). The limit of detection was estimated to be 8 fmol L−1, which is at least one order of magnitude lower than those achievable by mass spectrometry and spectroscopy (Table 1). The lower detection limit can be attributed to the multiplex amplification of AuNPs and the high turnover frequency of ALP.19 Moreover, this value is comparable to (or even lower than) those achievable using other electrochemical strategies for detection of glycoproteins including PSA (1.6 pg mL−1),17 sialylated glycoproteins fetuin (0.33 fM) and asialofetuin (0.54 fM),39 lactoferrin (145 pg mL−1),40 α-fetoprotein (4 pg mL−1),41 and ovalbumin (0.83 pg mL−1).42


Amplified voltammetric detection of glycoproteins using 4-mercaptophenylboronic acid/biotin-modified multifunctional gold nanoparticles as labels.

Liu L, Xing Y, Zhang H, Liu R, Liu H, Xia N - Int J Nanomedicine (2014)

(A) Differential pulse voltammograms for the detection of different concentrations of rHuEPO. (B) Plots of the Ipa against the rHuEPO concentration (0.02–10 pmol L−1). The inset shows the linear plots at concentrations of 0.02, 0.2, 0.5, 1.0, and 2.0 pmol L−1.Note: Each point was averaged from three replicates, and the error bars show the absolute standard.Abbreviation: rHuEPO, recombinant human erythropoietin.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4043723&req=5

f4-ijn-9-2619: (A) Differential pulse voltammograms for the detection of different concentrations of rHuEPO. (B) Plots of the Ipa against the rHuEPO concentration (0.02–10 pmol L−1). The inset shows the linear plots at concentrations of 0.02, 0.2, 0.5, 1.0, and 2.0 pmol L−1.Note: Each point was averaged from three replicates, and the error bars show the absolute standard.Abbreviation: rHuEPO, recombinant human erythropoietin.
Mentions: Differential pulse voltammetry is more sensitive than cyclic voltammetry, since it can decrease the background charging currents. Furthermore, we assessed the linear range, detection limit, and selectivity of this method with differential pulse voltammetry. The differential pulse voltammograms obtained at different rHuEPO concentrations are shown in Figure 4A. As a result, we found that the current increased linearly with the rHuEPO concentration in the range of 0.02–2.0 pmol L−1 and began to level off beyond 2 pmol L−1 (Figure 4B). All the relative standard deviations were found to be below 9.5%. The linear regression equation was expressed as Ipa (μA) =0.038+0.14 [rHuEPO] (pmol L−1) (R2=0.99). The limit of detection was estimated to be 8 fmol L−1, which is at least one order of magnitude lower than those achievable by mass spectrometry and spectroscopy (Table 1). The lower detection limit can be attributed to the multiplex amplification of AuNPs and the high turnover frequency of ALP.19 Moreover, this value is comparable to (or even lower than) those achievable using other electrochemical strategies for detection of glycoproteins including PSA (1.6 pg mL−1),17 sialylated glycoproteins fetuin (0.33 fM) and asialofetuin (0.54 fM),39 lactoferrin (145 pg mL−1),40 α-fetoprotein (4 pg mL−1),41 and ovalbumin (0.83 pg mL−1).42

Bottom Line: The MBA-biotin-AuNPs facilitated the attachment of streptavidin-conjugated alkaline phosphatase for the production of electroactive p-aminophenol from p-aminophenyl phosphate substrate.A detection limit of 8 fmol L(-1) for rHuEPO detection was achieved.Other glycosylated and non-glycosylated proteins, such as horseradish peroxidase, prostate specific antigen, metallothionein, streptavidin, and thrombin showed no interference in the detection assay.

View Article: PubMed Central - PubMed

Affiliation: College of Chemistry and Chemical Engineering, Anyang Normal University, Anyang, Henan, People's Republic of China ; College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan, People's Republic of China.

ABSTRACT
Ultrasensitive detection of protein biomarkers is essential for early diagnosis and therapy of many diseases. Glycoproteins, differing from other types of proteins, contain carbohydrate moieties in the oligosaccharide chains. Boronic acid can form boronate ester covalent bonds with diol-containing species. Herein, we present a sensitive and cost-effective electrochemical method for glycoprotein detection using 4-mercaptophenylboronic acid (MBA)/biotin-modified gold nanoparticles (AuNPs) (MBA-biotin-AuNPs) as labels. To demonstrate the feasibility and sensitivity of this method, recombinant human erythropoietin (rHuEPO) was tested as a model analyte. Specifically, rHuEPO was captured by the anti-rHuEPO aptamer-covered electrode and then derivatized with MBA-biotin-AuNPs through the boronic acid-carbohydrate interaction. The MBA-biotin-AuNPs facilitated the attachment of streptavidin-conjugated alkaline phosphatase for the production of electroactive p-aminophenol from p-aminophenyl phosphate substrate. A detection limit of 8 fmol L(-1) for rHuEPO detection was achieved. Other glycosylated and non-glycosylated proteins, such as horseradish peroxidase, prostate specific antigen, metallothionein, streptavidin, and thrombin showed no interference in the detection assay.

Show MeSH