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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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Selectivity and interference of the sensing protocol. 1, horseradish peroxidase; 2, prostate-specific antigen; 3, metallothionein; 4, streptavidin; 5, thrombin; 6, rHuEPO in buffer; 7, rHuEPO in buffer containing the five interfering proteins; 8, rHuEPO in serum. The concentration of rHuEPO was 1 pmol L−1Abbreviation: rHuEPO, recombinant human erythropoietin.
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f5-ijn-9-2619: Selectivity and interference of the sensing protocol. 1, horseradish peroxidase; 2, prostate-specific antigen; 3, metallothionein; 4, streptavidin; 5, thrombin; 6, rHuEPO in buffer; 7, rHuEPO in buffer containing the five interfering proteins; 8, rHuEPO in serum. The concentration of rHuEPO was 1 pmol L−1Abbreviation: rHuEPO, recombinant human erythropoietin.

Mentions: To demonstrate the selectivity of this sensor, we tested other proteins (glycoproteins and nonglycoproteins), such as horseradish peroxidase, PSA, metallothionein, SA, and thrombin, at a concentration ten times higher than that of rHuEPO. As shown in Figure 5, the negligible currents in these cases are indicative of the high selectivity of the method to rHuEPO. Furthermore, to investigate the interference, the sensor electrode was incubated with the rHuEPO solution containing above tested five interfering proteins. As a result, no apparent difference in the current was observed in the cases of absence and presence of these interferences, further verifying that the aptamer was highly specific to rHuEPO. Moreover, in the spectrum analysis, the protein detection is usually disturbed by the strong light scattering effect of serum. To demonstrate the viability of the sensor in biomedical samples, we examined its performance in the solution of blood serum. As a result, the current in 10% serum was the same as in the buffer, demonstrating that a serum environment shows no interference in the detection assay. Since phenylboronic acid can react with various carbohydrates, phenylboronic acid-based materials have been widely used in the selective separation of glycoproteins such as lactoferrin, horseradish peroxidase, ribonuclease B, α-acid glycoprotein, α-fetoprotein, and ovalbumin.21 We believe that the strategy developed here would also be suitable for the sensitive and selective detection of these glycoproteins on electrodes covered with the matched capture probes. Moreover, compared with mass spectrometry and spectroscopy, our method is simple, selective, and cost-effective. For example, in the mass spectrometric assay, sample cleanup and preconcentration (eg, analyte extraction and enrichment by immunoaffinity separation, with antibodies and dual digestion by trypsin as well as peptide-N/O-glycosidase) is required considering the complex sample matrix and low concentrations of glycoproteins in biological fluids.46


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)

Selectivity and interference of the sensing protocol. 1, horseradish peroxidase; 2, prostate-specific antigen; 3, metallothionein; 4, streptavidin; 5, thrombin; 6, rHuEPO in buffer; 7, rHuEPO in buffer containing the five interfering proteins; 8, rHuEPO in serum. The concentration of rHuEPO was 1 pmol L−1Abbreviation: rHuEPO, recombinant human erythropoietin.
© Copyright Policy
Related In: Results  -  Collection

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

f5-ijn-9-2619: Selectivity and interference of the sensing protocol. 1, horseradish peroxidase; 2, prostate-specific antigen; 3, metallothionein; 4, streptavidin; 5, thrombin; 6, rHuEPO in buffer; 7, rHuEPO in buffer containing the five interfering proteins; 8, rHuEPO in serum. The concentration of rHuEPO was 1 pmol L−1Abbreviation: rHuEPO, recombinant human erythropoietin.
Mentions: To demonstrate the selectivity of this sensor, we tested other proteins (glycoproteins and nonglycoproteins), such as horseradish peroxidase, PSA, metallothionein, SA, and thrombin, at a concentration ten times higher than that of rHuEPO. As shown in Figure 5, the negligible currents in these cases are indicative of the high selectivity of the method to rHuEPO. Furthermore, to investigate the interference, the sensor electrode was incubated with the rHuEPO solution containing above tested five interfering proteins. As a result, no apparent difference in the current was observed in the cases of absence and presence of these interferences, further verifying that the aptamer was highly specific to rHuEPO. Moreover, in the spectrum analysis, the protein detection is usually disturbed by the strong light scattering effect of serum. To demonstrate the viability of the sensor in biomedical samples, we examined its performance in the solution of blood serum. As a result, the current in 10% serum was the same as in the buffer, demonstrating that a serum environment shows no interference in the detection assay. Since phenylboronic acid can react with various carbohydrates, phenylboronic acid-based materials have been widely used in the selective separation of glycoproteins such as lactoferrin, horseradish peroxidase, ribonuclease B, α-acid glycoprotein, α-fetoprotein, and ovalbumin.21 We believe that the strategy developed here would also be suitable for the sensitive and selective detection of these glycoproteins on electrodes covered with the matched capture probes. Moreover, compared with mass spectrometry and spectroscopy, our method is simple, selective, and cost-effective. For example, in the mass spectrometric assay, sample cleanup and preconcentration (eg, analyte extraction and enrichment by immunoaffinity separation, with antibodies and dual digestion by trypsin as well as peptide-N/O-glycosidase) is required considering the complex sample matrix and low concentrations of glycoproteins in biological fluids.46

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