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SERS-based quantitative detection of ovarian cancer prognostic factor haptoglobin.

ABSTRACT: Surface-enhanced Raman spectroscopy (SERS) is increasingly being used for biosensing because of its high sensitivity and low detection limit, which are made possible by the unique Raman 'fingerprint' spectra from the biomolecules. Here we propose a novel SERS method for the fast, sensitive, and reliable quantitative analysis of haptoglobin (Hp), an acute phase plasma glycoprotein that is widely gaining application as a prognostic ovarian cancer biomarker. We exploited the peroxidase activity of the hemoglobin-haptoglobin (Hb-Hp) complex formed by the selective and specific binding of Hp to free Hb to catalyze the reaction of 3,3',5,5'-tetramethylbenzidine (TMB) substrate and hydrogen peroxide to result in the final product of strongly SERS-active TMB(2+). We observed a linear increase in the SERS signal of TMB(2+) with increasing concentrations of Hb-Hp complex from 50 nM to 34 ?M. Based on this concentration-dependent SERS spectrum, we quantified Hp in clinical samples. We observed that our inference about the prognosis of the disease coincided with the histology data and that our method was much more sensitive than the enzyme-linked immunosorbent assay method.

SUBMITTER: Perumal J

PROVIDER: S-EPMC4358420 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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SERS-based quantitative detection of ovarian cancer prognostic factor haptoglobin.

Perumal Jayakumar J Balasundaram Ghayathri G Mahyuddin Aniza P AP Choolani Mahesh M Olivo Malini M

International journal of nanomedicine 20150306

Surface-enhanced Raman spectroscopy (SERS) is increasingly being used for biosensing because of its high sensitivity and low detection limit, which are made possible by the unique Raman 'fingerprint' spectra from the biomolecules. Here we propose a novel SERS method for the fast, sensitive, and reliable quantitative analysis of haptoglobin (Hp), an acute phase plasma glycoprotein that is widely gaining application as a prognostic ovarian cancer biomarker. We exploited the peroxidase activity of ...[more]

PMID: 25834423

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Project description:Screening for specific biomarkers of early-stage detection of ovarian cancer is a major health priority due to the asymptomatic nature and poor survival characteristic of the disease. We utilised two-dimensional gel electrophoresis (2DE) to identify differentially expressed proteins in the serum of ovarian cancer patients that may be useful as biomarkers of this disease. In this study, 38 ovarian cancer patients at different pathological grades (grade 1 (n=6), grade 2 (n=8) and grade 3 (n=24)) were compared to a control group of eight healthy women. Serum samples were treated with a mixture of Affigel-Blue and protein A (5 : 1) for 1 h to remove high abundance protein (e.g. immunoglobulin and albumin) and were displayed using 11 cm, pH 4-7 isoelectric focusing strips for the first dimension and 10% acrylamide gel electrophoresis for the second dimension. Protein spots were visualised by SYPRO-Ruby staining, imaged by FX-imager and compared and analysed by PDQuest software. A total of 24 serum proteins were differentially expressed in grade 1 (P<0.05), 31 in grade 2 (P<0.05) and 25 in grade 3 (P<0.05) ovarian cancer patients. Six of the protein spots that were significantly upregulated in all groups of ovarian cancer patients were identified by nano-electrospray quadrupole quadrupole time-of-flight mass spectrometry (n-ESIQ(q)TOFMS) and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOFMS) as isoforms of haptoglobin-1 precursor (HAP1), a liver glycoprotein present in human serum. Further identification of the spots at different pathological grades was confirmed by Western blotting using monoclonal antibody against a haptoglobin epitope contained within HAP1. Immunohistochemical localisation of HAP1-like activity was present in malignant ovarian epithelium and stroma but strong immunostaining was present in blood vessels, areas with myxomatous stroma and vascular spaces. No tissue localisation of HAP1-like immunoreactivity was observed in normal ovarian surface epithelium. These data highlight the need to assess circulating concentration of HAP1 in the serum of ovarian cancer patients and evaluate its potential as a biomarker in the early diagnosis of ovarian cancer.

Dataset Information

SERS-based quantitative detection of ovarian cancer prognostic factor haptoglobin.

Publications

SERS-based quantitative detection of ovarian cancer prognostic factor haptoglobin.

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