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Dec 1, 2010 (Vol. 30, No. 21)

Multiplexing Finds Diverse Applications

Non-PCR Point-of-Care Diagnostics and Digital Assays Draw the Attention of Research Scientists

  • Assays Go Digital

    Click Image To Enlarge +
    BioCode-1000 Analyzer is a barcoded magnetic bead optical imaging system for a 96-well microplate format. After undergoing reaction, the beads settle to the bottom of each well, allowing for barcode identification and fluorescent signal detection. Multiple analyte tests can be performed in each well. [Applied Biocode]

    Applied Biocode offers a barcoded, magnetic-bead, high-throughput approach based on digital technology, according to company president, Winston Ho, Ph.D., who was one of the speakers at the “Biodetection Technologies” meeting.

    “The motivation for development of our platform was the need to process a large number of tests—up to 1,024—in a single microwell.”

    The barcodes the company uses are similar to those found in other commercial operations. The number of permutations of arrangements of the barcode is 2N, so that by increasing the number of wide and narrow bars in a module from 1 to 10, the number of unique identifiers can be easily extended. 

    The company’s technology employs a permanent digital barcode, which is bonded to the beads using photolithography for greater batch consistency. It is open ended, scalable, and the detection of the signal is accomplished with an LED and a lamp, rather than through the use of lasers and flow cytometers, thus providing greater robustness, according to Dr. Ho.

    Because of the elevated accuracy of the barcode classification, a large number of different beads can be loaded in a single microwell. Positive reactions are detected using fluorescence with the dye, such as phycoerythrin, which fluoresces at 575 nm.

    By combining digital barcodes with molecular diagnostics, the company has built a polymer bead with the magnetic barcodes encapsulated inside and permanently encoded. This accuracy allows up to 1,024 tests to be combined in a single tube.

    Applied Biocode is in collaboration with a number of partners to develop various DNA, RNA, and protein assays. These include IL-2, with a sensitivity down to the pictogram range, MRSA, and a ten-plex panel for cytokines. 

    GE Global Research (ge.geglobalresearch.com) has developed a lead technology platform for measuring multiple biomarkers in tumor tissues, explained John Burczak, Ph.D., chief scientist for molecular imaging at the company. His team is evaluating methods for the detection of cancerous pathology in tissue samples.

    The conventional approach, when suspected malignant tissues are scored, is by a somewhat subjective judgment of the pathologist, who ranks the tissues on a numerical scale according to the degree of malignancy. The decision is frequently controverted by alternative expert opinion, and in some cases by the original evaluator on re-examination.

  • Multiple Biomarker Analysis

    A more objective approach is the use of antibodies directed against cancer-associated proteins, reacted with tissue homogenates, but the strategy only provides an average over the entire sample, rather than revealing tiny microfoci of cancerous tissue within. Given that pathology samples are extremely heterogeneous, containing necrotic, normal, and transformed cells, this method may miss important features of the test material.

    The approach adopted by Dr. Burczak’s team is based on the use of tumor biomarkers detected by immunocytochemistry. Four different antibodies are coupled to four different fluorescent dyes. The tumor slices are stained and the image is quantified using digital microscopy. The dyes are quenched with a mild agent that does not damage the proteins, and the entire process is repeated. Using this serialized approach it is possible to follow up to 100 biomarkers on the same slice of tissue, according to Dr. Burczak.

    “To avoid loss of information, we test the most fragile proteins first and then work up to the more robust markers that have no chance of sustaining damage by multiple quenching steps. Essentially, we can do what cell sorting does but on a solid tissue.”

    The approach also allows evaluation of the status of individual cells or even regions within the cells. By varying the level of magnification from 5x to 20x through 40x it is possible to visualize the abundance of proteins within the cytoplasm or other regions within the cell. “We are probing deeply into subcellular quantitative analysis using tissue slices,” reported Dr. Burczak. 

    As an example of the platform’s applications, he mentioned the Her-2 neu protein, the estrogen receptor critical for binding of the antitumor agent tamoxifen. Tumors that do not have estrogen receptors do not respond to such therapies, and prior to the development of these detection systems it would not have been possible to decide on the appropriate course of treatment for breast cancer patients.

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