Simplified multiplex biomarker testing using flow-through microarray technology

Biomarker testing is an essential component of contemporary personalized medicine that has become a routine part of everyday healthcare. In its simplest forms, biomarker testing can be performed at home using devices such as lateral flow pregnancy tests, which measure the level of human chorionic gonadotropin (hCG) in urine. More complex biomarker assays that require higher levels of sensitivity, intricate procedures, and numerical data analysis are performed in specialized laboratories with more sophisticated instrumentation. Examples include tests that can detect mutations in the BRCA1/2 genes for surveillance and prevention of breast and ovarian cancers, and the measurement of HER2/neu over-expression in breast and gastric cancers for disease characterization and prediction of response to trastuzumab (Herceptin) and lapatinib (Tykerb).^1,2 

 Recent advances in genomics, proteomics, and metabolomics have led to significant biomarker discoveries and the development of novel tests for more effective disease diagnostics and management.^1,2 Due to the complexity of biological systems and the wide variance in individual genetic and metabolic backgrounds, tests based on single biomarkers may perform with low sensitivity and/or specificity. Thus, there is a trend toward the development of tests based on the simultaneous measurement of multiple biomarkers. These tests have shown significant improvement in assay performance and clinical utility. 

A number of multiplex technologies are currently available for biomarker testing, including suspension microspheres, planar microarrays, microplate-based microarrays, three dimensional gels, and LCMS.^2-5 While each approach provides distinct advantages for multiplexed biomarker analysis, no technology presently offers a combination of high analyte sensitivity, moderate to high multiplexing capability, short time to results, and simplicity for use in near-patient settings.³

A recently developed multiplex technology based on the use of flow-through microarrays significantly simplifies the assay process and promises to bring the clinical benefits of multiplexed biomarker analysis to near-patient settings for tests where rapid results and high sensitivity are required. This technology utilizes highly porous silicon chips (over 200,000 of 10×10×450 micron pores) on which more than 500 features can be spotted with a microarray printer (Figure 1A).

The printed capture molecules (e.g., antibodies) efficiently adsorb to all surfaces of the microchannels, significantly increasing the assay surface area compared to similarly formatted planar arrays (Figure 1B). Assays are performed in an instrument that automates the movement of the chips into different wells in 96-well plates containing samples, assay reagents, and wash buffers. The instrument repeatedly draws up and expels the contents of each well through the pores of the chip using user-defined settings for mixing frequency and duration. Specific analytes that are captured on the individual spots on the chips are detected using a horseradish peroxidise-luminol system to generate chemiluminescent signals that can be quantitated with an integrated CCD camera (Figure 1C). The ability to flow assay reagents back and forth through the porous silicon chip results in conditions approaching those of a homogeneous reaction. Vigorous mixing substantially enhances the kinetics of interaction of assay reagents with the immobilized proteins on the chip surface, and improves the removal of non-
specifically bound molecules during washes. This improves sensitivity and significantly reduces overall assay times for routine sandwich immunoassays. The system is capable of parallel processing of up to eight test samples and is compatible with a wide range of sample types including serum/plasma, CSF, and dried blood spots. 

The flow-through microarray technology has recently been evaluated for use in several nucleic acid⁶ and protein multiplexed assays focussed on rapid, near-patient applications. Below we describe three protein-based tests that are currently in development.

Abusive head trauma (AHT) in infants can be difficult to diagnose in an emergency room setting because they may not exhibit overt signs of brain injury and are unable to provide a verbal history.^7-8 A panel of serum-based protein biomarkers were measured simultaneously using flow-through microarrays to rapidly screen infants with suspected AHT to determine if a head CT scan was required. The test shows promising performance in stratifying infants with and without AHT. Implementation of this test may lead to a reduction in unnecessary head CTs and thus the long-term risks associated with high dose x-ray exposure in these patients.

This technology has also been used to develop screening tests for suspected parasitic infections. Crude extract and recombinant proteins from various parasites were printed on the porous silicon chips, and patient sera were analysed for IgG against the immobilized parasite proteins. In acute care settings, patients presenting with non-specific symptoms (e.g., gastrointestinal discomfort) and suspected of having a parasitic infection can be rapidly screened to identify the causative species and determine the proper course of treatment.⁹ This test is particularly advantageous in cases where patients exhibit co-infection with more than one parasite. 

Finally, the detection of allergen-specific IgE in blood has been routinely used as a non-invasive method of diagnosing allergies.^10,11 Drawbacks to this approach include the inability to concurrently screen large numbers of allergens and the long time-to-results, typically requiring multiple visits to the allergist’s office for follow-up and clinical management. Moreover, with a trend toward component-resolved diagnosis in allergy testing, there is a need for higher multiplexing capabilities.^12,13 The flow-through technology has been used to develop assays against multiple proteins from an allergen. With allergy-positive patients generally displaying different patterns of responses to the various proteins, this test may provide better characterization of the allergy in terms of severity, cross-reactivity, or the likelihood of outgrowing allergies in children. The rapid time-to-answer of this multiplexed allergy assay opens the possibility of completing broad screening, diagnosis, and treatment decisions in a single doctor’s office visit.

Multiplexed biomarker testing benefits patients by providing the comprehensive analysis necessary to enable personalized treatment. This can result in more efficient treatment approaches, delivering significant cost savings to the healthcare system. Moving multiplexed biomarker analysis from specialized central laboratories to the doctor’s office or emergency room can extend these benefits to a broad range of acute and chronic disease states. The flow-through microarray technology represents a novel approach that can meet this challenge and bring multiplexed biomarker testing closer to the patient. 

References 

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