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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) Electrochemical impedance spectroscopy of Au (curve a), Au/aptamers (curve b), Au/aptamers/MCH/BSA (curve c), Au/aptamers/MCH/BSA/rHuEPO (curve d), and Au/aptamers/MCH/BSA/rHuEPO/MBA-biotin-AuNPs (curve e) electrodes in [Fe(CN)6 ]3−/4−. (B) Cyclic voltammograms of the aptamer-covered electrode with (black curve) and without (red curve) the capture of rHuEPO, followed by the attachment of MBA-biotin-AuNPs as well as SA-ALP for the generation of p-AP. Blue curve corresponds to that after the capture of rHuEPO and the treatment with biotin-AuNPs in place of MBA-biotin-AuNPs. The concentration of rHuEPO was 10 pmol L−1. The arrow indicates the scan direction.Abbreviations: biotin-AuNP, biotin-modified gold nanoparticle; BSA, bovine serum albumin; MBA-biotin-AuNP, 4-mercaptophenylboronic acid/biotin-modified gold nanoparticle; MCH, 6-mercapto-1-hexanol; p-AP, p-aminophenol; Q, a constant phase element; Ret, electron-transfer resistance; rHuEPO, recombinant human erythropoietin; Rs, resistance between working and reference electrodes; SA-ALP, streptavidin-conjugated alkaline phosphatase; Zw, Warburg impedance.
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f3-ijn-9-2619: (A) Electrochemical impedance spectroscopy of Au (curve a), Au/aptamers (curve b), Au/aptamers/MCH/BSA (curve c), Au/aptamers/MCH/BSA/rHuEPO (curve d), and Au/aptamers/MCH/BSA/rHuEPO/MBA-biotin-AuNPs (curve e) electrodes in [Fe(CN)6 ]3−/4−. (B) Cyclic voltammograms of the aptamer-covered electrode with (black curve) and without (red curve) the capture of rHuEPO, followed by the attachment of MBA-biotin-AuNPs as well as SA-ALP for the generation of p-AP. Blue curve corresponds to that after the capture of rHuEPO and the treatment with biotin-AuNPs in place of MBA-biotin-AuNPs. The concentration of rHuEPO was 10 pmol L−1. The arrow indicates the scan direction.Abbreviations: biotin-AuNP, biotin-modified gold nanoparticle; BSA, bovine serum albumin; MBA-biotin-AuNP, 4-mercaptophenylboronic acid/biotin-modified gold nanoparticle; MCH, 6-mercapto-1-hexanol; p-AP, p-aminophenol; Q, a constant phase element; Ret, electron-transfer resistance; rHuEPO, recombinant human erythropoietin; Rs, resistance between working and reference electrodes; SA-ALP, streptavidin-conjugated alkaline phosphatase; Zw, Warburg impedance.

Mentions: Electrochemical impedance spectroscopy has been used to examine the property of self-assembled monolayers, including surface coverage and monolayer composition, and the conduction ability of the modified electrode. Herein, the immobilization of the aptamers and the attachment of rHuEPO and modified gold nanoparticles were characterized by electrochemical impedance spectroscopy. A modified Randles equivalent circuit was used to fit the impedance spectra and to determine electrical parameters for each step. As shown in the inset of Figure 3A, the circuit included Rs (the electrolyte resistance between working and reference electrodes), Zw (the Warburg impedance), Q (a constant phase element representing the double layer capacitance for an unmodified electrode or the capacitance of the self-assembled monolayers for the modified electrodes) and Ret (the electron-transfer resistance). As shown in Figure 3A, the bare gold electrode showed a small electron transfer resistance (curve a), demonstrating a fast electron transfer process. The increase in the Ret implied the immobilization of anti-rHuEPO aptamers (curve b), and the result can be explained by the fact that the negatively charged aptamers repulsed the negatively charged [Fe(CN)6]3−/4− from the sensor surface. The modification of Au/aptamers with MCH and BSA resulted in an increase in the Ret (curve c). The result is understandable, since the block of unreacted gold surface by the MCH and BSA could retard the interfacial electron transfer kinetics of [Fe(CN)6]3−/4− anions. Interestingly, we found that incubation of the aptamer-modified electrode with rHuEPO solution led to a small increase in the Ret (curve d), indicating that rHuEPO was captured by the sensing electrode. In turn, we found that the charge-transfer impedance decreased with the attachment of MBA-biotin-AuNPs (curve e), which was attributed to the high conducting capability of AuNPs.


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) Electrochemical impedance spectroscopy of Au (curve a), Au/aptamers (curve b), Au/aptamers/MCH/BSA (curve c), Au/aptamers/MCH/BSA/rHuEPO (curve d), and Au/aptamers/MCH/BSA/rHuEPO/MBA-biotin-AuNPs (curve e) electrodes in [Fe(CN)6 ]3−/4−. (B) Cyclic voltammograms of the aptamer-covered electrode with (black curve) and without (red curve) the capture of rHuEPO, followed by the attachment of MBA-biotin-AuNPs as well as SA-ALP for the generation of p-AP. Blue curve corresponds to that after the capture of rHuEPO and the treatment with biotin-AuNPs in place of MBA-biotin-AuNPs. The concentration of rHuEPO was 10 pmol L−1. The arrow indicates the scan direction.Abbreviations: biotin-AuNP, biotin-modified gold nanoparticle; BSA, bovine serum albumin; MBA-biotin-AuNP, 4-mercaptophenylboronic acid/biotin-modified gold nanoparticle; MCH, 6-mercapto-1-hexanol; p-AP, p-aminophenol; Q, a constant phase element; Ret, electron-transfer resistance; rHuEPO, recombinant human erythropoietin; Rs, resistance between working and reference electrodes; SA-ALP, streptavidin-conjugated alkaline phosphatase; Zw, Warburg impedance.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4043723&req=5

f3-ijn-9-2619: (A) Electrochemical impedance spectroscopy of Au (curve a), Au/aptamers (curve b), Au/aptamers/MCH/BSA (curve c), Au/aptamers/MCH/BSA/rHuEPO (curve d), and Au/aptamers/MCH/BSA/rHuEPO/MBA-biotin-AuNPs (curve e) electrodes in [Fe(CN)6 ]3−/4−. (B) Cyclic voltammograms of the aptamer-covered electrode with (black curve) and without (red curve) the capture of rHuEPO, followed by the attachment of MBA-biotin-AuNPs as well as SA-ALP for the generation of p-AP. Blue curve corresponds to that after the capture of rHuEPO and the treatment with biotin-AuNPs in place of MBA-biotin-AuNPs. The concentration of rHuEPO was 10 pmol L−1. The arrow indicates the scan direction.Abbreviations: biotin-AuNP, biotin-modified gold nanoparticle; BSA, bovine serum albumin; MBA-biotin-AuNP, 4-mercaptophenylboronic acid/biotin-modified gold nanoparticle; MCH, 6-mercapto-1-hexanol; p-AP, p-aminophenol; Q, a constant phase element; Ret, electron-transfer resistance; rHuEPO, recombinant human erythropoietin; Rs, resistance between working and reference electrodes; SA-ALP, streptavidin-conjugated alkaline phosphatase; Zw, Warburg impedance.
Mentions: Electrochemical impedance spectroscopy has been used to examine the property of self-assembled monolayers, including surface coverage and monolayer composition, and the conduction ability of the modified electrode. Herein, the immobilization of the aptamers and the attachment of rHuEPO and modified gold nanoparticles were characterized by electrochemical impedance spectroscopy. A modified Randles equivalent circuit was used to fit the impedance spectra and to determine electrical parameters for each step. As shown in the inset of Figure 3A, the circuit included Rs (the electrolyte resistance between working and reference electrodes), Zw (the Warburg impedance), Q (a constant phase element representing the double layer capacitance for an unmodified electrode or the capacitance of the self-assembled monolayers for the modified electrodes) and Ret (the electron-transfer resistance). As shown in Figure 3A, the bare gold electrode showed a small electron transfer resistance (curve a), demonstrating a fast electron transfer process. The increase in the Ret implied the immobilization of anti-rHuEPO aptamers (curve b), and the result can be explained by the fact that the negatively charged aptamers repulsed the negatively charged [Fe(CN)6]3−/4− from the sensor surface. The modification of Au/aptamers with MCH and BSA resulted in an increase in the Ret (curve c). The result is understandable, since the block of unreacted gold surface by the MCH and BSA could retard the interfacial electron transfer kinetics of [Fe(CN)6]3−/4− anions. Interestingly, we found that incubation of the aptamer-modified electrode with rHuEPO solution led to a small increase in the Ret (curve d), indicating that rHuEPO was captured by the sensing electrode. In turn, we found that the charge-transfer impedance decreased with the attachment of MBA-biotin-AuNPs (curve e), which was attributed to the high conducting capability of AuNPs.

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
Related in: MedlinePlus