May 15, 2012 (Vol. 32, No. 10)

Array-Based Assay System Facilitates Nanoscale Protein Studies

Life scientists are under increasing pressure to do more with less, with a particular need to generate more highly multiplexed proteomic data from the smallest of sample sizes in less and less time. Unfortunately, many conventional protein analysis platforms have drawbacks that make them less than ideal.

Traditional protein microarray technologies often print inconsistent features, leading to poor sensitivity and reproducibility and the inability to detect proteins present in small quantities of sample. In addition, many protein analysis assays require large amounts of sample material and reagents and can exhibit slow reaction kinetics.

NanoInk® has developed a new generation of array-based instrument systems, assay kits, and contract services for nanoscale protein studies. Based on patented Dip Pen Nanolithography® (DPN®) technology, NanoInk’s platform is capable of creating multiplexed protein arrays for use in miniaturized immunoassays. NanoInk assays consume smaller amounts of sample and reagents than traditional assays, generating the desired data with less starting material.

At the heart of the NanoInk solution is the NanoArray Assay System™ (Figure 1), an integrated instrument that couples nanoscale array deposition techniques with the latest generation of fluorescent detection technologies. The system can create highly reproducible protein arrays over millimeter areas with nanometer precision and then fluorescently image these nanoarrays at 0.5 μm resolution.

These proteomic assays require as little as 2 μL of sample to identify, detect, and quantitate clinically and biologically relevant, low abundance proteins from a wide variety of sample types in applications such as biomarker analysis, translational medicine, and toxicology.

Configured nanoscale assay kits are available for simultaneously evaluating a panel of 10 human cytokines implicated in inflammation in a single sub-array. NanoInk has also conducted studies validating the nanoarray platform’s utility for multiplexed analysis in a variety of assays including human inflammation and angiogenesis, as well as rodent renal toxicity.

Figure 1. The NanoArray Assay System is composed of the NanoArrayer 3000 and the NanoScan 900. The NanoArrayer 3000 is capable of patterning thousands of features into each sub-array. A confocal scanner with two sensitive photomultipliers, the NanoScan 900 can detect fluorescence across an entire arrayed surface.

Benefits of Miniaturization

Small sample volume requirements are important because many biological samples are available in such tiny quantities that they cannot be analyzed using conventional platforms. As an example, traditional protein analysis platforms require 50–100 µL of sample; these sample volumes are not always attainable. Because NanoInk’s multiplexed nanoarray-based assays require just 2 µL of sample, they can successfully analyze multiple protein biomarkers within even rare and hard-to-collect samples such as rodent serum and urine, cerebrospinal fluid, human tumor biopsies, tears, and dried blood spots.

The ultra-low sample requirement and assay volume of nanoscale assays also translates to substantially lower reagent usage compared to traditional assays.

The NanoArray Assay System can simultaneously deposit multiple capture antibodies on a slide to create a unique immunoassay platform. Printed antibody features exhibit highly uniform spot morphology, so multiplexed nanoarray immunoassays can achieve femtograms/mL sensitivity with 2 μL of sample, comparable to or better than the sensitivities of more traditional assay platforms that consume 50 μL or more of sample.

Even when generous sample volumes are available, biomarker concentrations often fall below common limits of detection. With femtogram-level sensitivity, the NanoInk platform can detect even low-abundance biomarkers from most clinical samples.

Miniaturization also contributes to improved assay throughput. By depositing thousands of uniform features in the area occupied by just one spot on a conventional protein microarray, the NanoInk platform can pattern as many as 96 sub-arrays on one nanoarray slide. This small scale and precision enables highly multiplexed protein analysis on a single slide.

Nanoarray assays are also SBS-compliant and compatible with most commercially available lab automation systems, providing the choice of automated high-throughput proteomic analysis or simple benchtop testing using standard immunoassay protocols. Both formats deliver rapid-reaction kinetics within miniaturized reagent volumes.

Case Study: Measuring Biomarkers in Dried Blood Spot Samples

There has been a recent upsurge in interest for using dried blood spot (DBS) samples for the clinical measurement of biomarkers. DBS technology was implemented clinically in the 1970s for neonatal screening and has since been expanded to many other applications.

Benefits of DBS sampling include a small blood volume requirement, easy sample collection, no centrifugation steps, and simplified storage and transport. However, the sensitivity of DBS tests is often poor, and most assay formats that use DBS samples are tedious and have limited throughput. Sensitivity and sample-size issues have, until now, limited the utility of DBS sampling for measuring low-abundance biomarkers.

NanoInk has combined the benefits of a 2 µL sample size requirement with a DBS elution protocol to develop a miniaturized immunoassay capable of measuring multiple biomarkers in small-volume samples with improved sensitivity. A multiplexed cytokine DBS assay has been fully validated and shown to have low well-to-well variability, low slide-to-slide variability, and excellent standard and QC recoveries.

For this validation study, the NanoArrayer 3000 was used to print cytokine capture antibodies in 48 different sub-arrays of a functionalized glass slide. The resulting feature sizes were between 2.5–3 µm in diameter.

DBS standards and QC samples were eluted in an elution buffer prior to analysis in the multiplex NanoArray assay (Figure 2) using streptavidin-Alexa Fluor® 647 as a detection molecule. DBS accuracy and precision validation statistics were determined for three different runs over two days. The multiplexed cytokine DBS assay passed all validation criteria.

In addition, NanoInk has conducted preliminary correlation studies with DBS and serum samples from rheumatoid arthritis patients for a selection of biomarkers (including TNF-α, leptin, MIP-1b, IP-10, IL-8, and IFNγ). The DBS and serum samples collected thus far have been tested in a nanoarray multiplex assay, and the concentrations of biomarkers were determined. This study is still under way, but preliminary results suggest a significant DBS-serum correlation for a panel of biomarkers.

This case study has confirmed that DBS sampling can provide significant benefits over conventional blood draws. The nanoarray assay has been validated using DBS samples in a multiplex format, and we have demonstrated that the NanoInk assay can provide a significant amount of biomarker data with minimum sample volumes.

Figure 2. Nanoscale sandwich ELISA with fluorescent molecule detection

Haris Jamil, Ph.D. ([email protected]), is director, Nano BioDiscovery Laboratory, NanoInk.

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