The emergence of personalized medicine is continuing to drive interest in discovering novel biomarkers to effectively diagnose, monitor, and treat diseases based on an individual’s unique molecular signature. Over the past couple of decades, the use of DNA microarrays has led to the discovery of numerous disease-specific gene signatures.1 Likewise, proteomic approaches such as mass spectrometry or two-dimensional gel electrophoresis have identified an abundance of protein biomarkers.2,3 Multivariate analyses of these markers yield panels or signatures that can provide better predictive value over single markers. One of the major challenges in translating these discoveries from the bench to clinical utility is validating their performance in properly controlled, larger-scale studies. Due to the cost and complexity of most discovery platforms, biomarker panel validation is typically performed on alternative platforms that are better suited for routine testing. Important requirements for validation platforms include the following:
- The ability to multiplex the analysis of relevant analytes in a single assay
- A broad dynamic range to test for analytes that may be present in widely different concentrations
- High detection sensitivity for the measurement of low abundant markers and, in special cases, to allow the use of low sample volumes to conserve scarce clinical samples
- Short time to results to increase overall throughput and make diagnostic implementation more practical
- Automation and ease of use in a standard clinical laboratory setting.
Flow-through microarrays represent a novel approach to simplifying biomarker testing by addressing each of these requirements.4 The microarrays are comprised of 6.5 mm2 square chips mounted on plastic tubes (Figure 1). The chips are made of functionalized porous silicon with >200,000 microchannels, each measuring 10 x 10 x 450 μm. Capture molecules (e.g., amino-terminated oligonucleotides, protein antigens, or antibodies, etc.) are spotted on the chips using a microarray printer, resulting in their covalent linkage to the surface. One spot occupies approximately 70 microchannels. Each chip can be sized to accommodate between 16 and 576 features, allowing for a wide range of multiplex capabilities with replicates and controls. A highly sensitive chemiluminescent detection system is used for analyte imaging and quantitation. With this system, detection limits can be as low as attomolar concentrations for nucleic acids and proteins while the dynamic range can reach up to four log units. Precision is typically less than 10% CV for both proteins and nucleic acids.
Assays with the flow-through microarrays are easily automated using a robotic arm that moves the chips through a series of microplate wells that are filled with test samples, reagents and wash buffers (Figure 2). The system can run up to eight chips in parallel, one for each sample.
The principle advantage of this approach is the presence of flow-through microchannels. They allow samples and reagents to be passed back and forth through the microarray chip in close contact to the capture surface, significantly reducing incubation times compared to orbital shaking, carousel rotation, or laminar flow. For example, gene expression assays are completed in less than three hours, which includes oligonucleotide hybridization, washing steps, imaging of the chemiluminescent signals, and generating a final, custom report. By comparison, other gene expression systems often require at least 16 hours for the hybridization step alone. Protein assays on the flow-through system are typically completed in 60 to 90 minutes. The microchannels also permit vigorous washing of the chip after sample and reagent incubation, which greatly reduces the effects of nonspecific binding.
In order to show interplatform concordance between the flow-through system and other commercially available global gene expression platforms, a comparative study was performed using an established quality control protocol designed to evaluate their reproducibility.5 The overall results showed that the flow-through system has similar reproducibility, repeatability, and sensitivity to these other platforms while performing automated assays in a significantly shorter time, making it ideal for accurately translating information from genome wide expression arrays onto low density microarrays suitable for routine biomarker testing.6
Flow-through technology is currently being demonstrated as a translational platform in a multicenter, retrospective study to investigate a Risk of Recurrence Assay in breast cancer patients using archival FFPE tumor samples. The study was designed to evaluate the performance of the gene expression assay in predicting the five-year risk of distant metastasis as well as the ability of clinical laboratories to perform the test locally. The assay evaluates the expression levels of a select set of genes that can provide information on ER/PR/Her-2 status, molecular subtype, as well as data on proliferation and immune response pathways. This test is meant to provide oncologists with immediate information for predicting disease recurrence and selecting the proper treatments for certain groups of breast cancer patients. Overall, the use of the flow-through system in this study illustrates its ability to translate a complex gene expression assay into a relatively simple clinical test that can be offered at local laboratories.
One unique feature of the flow-through system is its ability to employ very small sample volumes as a result of its high sensitivity. For example, a multiplex protein assay has been developed that requires only 2 µL of serum for diagnosing traumatic brain injuries in newborn infants. By comparison, single analyte plate ELISA methods could use up to 50 µL of serum for measuring the same protein panel, while bead-based multiplex systems can require as much as 25 µL. Typical heal-sticks on newborn infants collect only a few hundred microliters of serum, limiting the number of assays that can be performed using traditional methods. However, the inherent sensitivity of the flow-through system permits multiple analyses on a variety of different biological sample types such as blood spots, saliva, or swabs.
Overall, the flow-through microarray system is designed to simplify multiplex biomarker assay validation and enable the transition from discovery to clinical testing. It is an “all-in-one” system that automates the entire assay, including data collection and report generation. Its simplicity allows it to be more widely implemented in non-central laboratory settings, and it enables faster assay development and higher throughput. Flow-through microarrays can accelerate the validation and acceptance of new clinical tests as tools for personalized medicine, leading to improved disease diagnosis and patient management.
About the Authors
- Van’t Veer LJ, Bernards R. Enabling personalized cancer medicine through analysis of gene-expression patterns. Nature. 2008;452:564-570.
- Kingsmore SF. Multiplexed protein measurement: technologies and applications of protein and antibody arrays. Nat Rev Drug Discov. 2006;5:310-320.
- Conrads TP, Zhou M, Petricoin EF, Liotta L, Veenstra TD. Cancer diagnosis using proteomic patterns. Expert Rev Mol Diagn. 2003;3:411-420.
- Cheek BJ, Steel AB, Torres MP, Yu Y-Y, Yang H. Chemiluminescence detection for hybridization assays on the flow-thru chip, a three dimensional microchannel biochip. Anal Chem. 2001;73:5777-5783.
- Shi L, Reid LH, Jones WD, et al. The Microarray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements. Nat Biotechnol. 2006;24:1151-1161.
- Jones M, Dempsey A, Englert D. Gene expression profiling on the Ziplex® System demonstrates high interplatform concordance. Axela Inc. Whitepaper 2007. http://www.axela.com/docs/literatures/58.Gene_Exp_Profiling_WP_2012.pdf. Accessed February 22, 2013.