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Direct and multiplex quantification of protein biomarkers in serum samples using an immuno-magnetic platform.

ABSTRACT: A direct and ultrasensitive multiplex assay using an immuno-magnetic platform has been developed for the quantification of trace amounts of circulating cancer-associated antigens in serum. The detection is based on the specific immuno-interactions among the target antigen, detection antibody and capture antibody that is immobilized on the surface of magnetic nanoparticles. The sandwiched immuno-assembly is then labelled with turn-on fluorophores and detected with a fluorescence imaging system. To afford a high signal-to-noise ratio, three turn-on fluorophores with unique optical properties have been designed and synthesized to label the target antigens. The developed assay has achieved a remarkable LOD down to the femto-molar regime without sample pre-treatment. This versatile assay can efficiently differentiate the target antigen from a protein matrix and simultaneously quantify multiple cancer-associated antigens, for instance, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), and prostate specific antigen (PSA) using only 6 ?L of serum sample in an hour. This novel system has a high applicability to serve as a universal and useful tool for early disease diagnostics.

SUBMITTER: Ho SL

PROVIDER: S-EPMC5477028 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Direct and multiplex quantification of protein biomarkers in serum samples using an immuno-magnetic platform.

Ho See-Lok SL Xu Di D Wong Man Shing MS Li Hung-Wing HW

Chemical science 20160104 4

A direct and ultrasensitive multiplex assay using an immuno-magnetic platform has been developed for the quantification of trace amounts of circulating cancer-associated antigens in serum. The detection is based on the specific immuno-interactions among the target antigen, detection antibody and capture antibody that is immobilized on the surface of magnetic nanoparticles. The sandwiched immuno-assembly is then labelled with turn-on fluorophores and detected with a fluorescence imaging system. T ...[more]

PMID: 28660042

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Project description:BACKGROUND:Currently, there are no FDA approved screening tools for detecting early stage ovarian cancer in the general population. Development of a biomarker-based assay for early detection would significantly improve the survival of ovarian cancer patients. METHODS:We used a multiplex approach to identify protein biomarkers for detecting early stage ovarian cancer. This new technology (Proseek® Multiplex Oncology Plates) can simultaneously measure the expression of 92 proteins in serum based on a proximity extension assay. We analyzed serum samples from 81 women representing healthy, benign pathology, early, and advanced stage serous ovarian cancer patients. RESULTS:Principle component analysis and unsupervised hierarchical clustering separated patients into cancer versus non-cancer subgroups. Data from the Proseek® plate for CA125 levels exhibited a strong correlation with current clinical assays for CA125 (correlation coefficient of 0.89, 95% CI 0.83, 0.93). CA125 and HE4 were present at very low levels in healthy controls and benign cases, while higher levels were found in early stage cases, with highest levels found in the advanced stage cases. Overall, significant trends were observed for 38 of the 92 proteins (p < 0.001), many of which are novel candidate serum biomarkers for ovarian cancer. The area under the ROC curve (AUC) for CA125 was 0.98 and the AUC for HE4 was 0.85 when comparing early stage ovarian cancer versus healthy controls. In total, 23 proteins had an estimated AUC of 0.7 or greater. Using a naïve Bayes classifier that combined 12 proteins, we improved the sensitivity corresponding to 95% specificity from 93 to 95% when compared to CA125 alone. Although small, a 2% increase would have a significant effect on the number of women correctly identified when screening a large population. CONCLUSIONS:These data demonstrate that the Proseek® technology can replicate the results established by conventional clinical assays for known biomarkers, identify new candidate biomarkers, and improve the sensitivity and specificity of CA125 alone. Additional studies using a larger cohort of patients will allow for validation of these biomarkers and lead to the development of a screening tool for detecting early stage ovarian cancer in the general population.

Project description:In the continuous combat against diseases, there is the need for tools that enable an improved diagnostic efficiency towards higher information density combined with reduced time-to-result and cost. Here, a novel fully integrated microfluidic platform, the Evalution™, is evaluated as a potential solution to this need. Encoded microparticles combined with channel-based microfluidics allow a fast, sensitive and simultaneous detection of several disease-related biomarkers. Since the binary code is represented by physically present holes, 210 different codes can be created that will not be altered by light or chemically induced degradation. Exploiting the unique features of this multiplex platform, hybridization chain reaction (HCR) is explored as a generic approach to reach the desired sensitivity. Compared to a non-amplified reference system, the sensitivity was drastically improved by a factor of 104, down to low fM LOD values. Depending on the HCR duration, the assay can be tuned for sensitivity or total assay time, as desired. The huge potential of this strategy was further demonstrated by the successful detection of a multiplex panel of six different nucleic acid targets including viruses and bacteria. The ability to not only discriminate these two categories but, with the same effort, also virus strains (human adenovirus and human bocavirus), virus subtypes (human adenovirus type B and D) and antibiotic-resistant bacteria (Streptococcus pneumonia), exemplifies the specificity of the developed approach. The effective, yet highly simplified, isothermal and protein-enzyme-free signal amplification tool reaches an LOD ranging from as low as 33 ± 4 to 151 ± 12 fM for the different targets. Moreover, direct detection in a clinically relevant sample matrix was verified, resulting in a detection limit of 309 ± 80 fM, approximating the low fM levels detectable with the gold standard analysis method, PCR, without the drawbacks related to protein enzymes, thermal cycling and elaborate sample preparation steps. The reported strategy can be directly transferred as a generic approach for the sensitive and specific detection of various target molecules in multiplex. In combination with the high-throughput capacity and reduced reagent consumption, the Evalution™ demonstrates immense potential in the next generation of diagnostic tools towards more personalized medicine.

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Direct and multiplex quantification of protein biomarkers in serum samples using an immuno-magnetic platform.

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Direct and multiplex quantification of protein biomarkers in serum samples using an immuno-magnetic platform.

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