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Miniaturization is the key to doing more with less: assay minimization is helping scientists keep up with day-to-day demands, as well as decreasing costs, increasing sensitivity, boosting throughput and enhancing precision.

From protein biomarker analysis to microstructure functionalization to cell biology studies, the pressure is on for life scientists to do more with less. We want more highly multiplexed proteomic data from the smallest of sample sizes, the ability to quickly functionalize smaller, denser sensor arrays with less material and higher content analytics from individual cells. Just as it has been in the communications and computing industries, miniaturization is the key for doing more with less in the life science industry. Leveraging Dip Pen Nanolithography (DPN) technology, a tip-based nanofabrication technique that can rapidly and easily create micro and nanoscale patterns from a variety of materials on a multitude of substrates, NanoInk's DPN family of systems is capable of delivering the benefits of miniaturization to these life science applications.

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When we miniaturize assays, we minimize sample volume, lower reagent cost, increase sensitivity, boost throughput and enhance precision. Small sample volume requirements are important because many biological samples are available in such tiny quantities that they cannot be analyzed using conventional analysis platforms. As an example, traditional ELISA and beadbased immunoassay platforms require 50 to 100 [micro]L of sample; life scientists are deprived of the critical proteomic information they need when these sample volumes are not attainable. Because NanoInk's multiplexed nanoarray-based assays require just 2 [micro]L of sample, they can successfully analyze multiple protein biomarkers within rare and hard-to-collect samples, such as rodent serum and urine, spinal fluid, human tumor extracts, tears and dried blood spots. Just as importantly, the ultra-low sample requirement and assay volume of nanoscale assays translates to substantially lower reagent costs compared to traditional assays.

Even when generous sample volumes are available, biomarker concentrations often fall below common limits of detection. Because DPN-deposited nanopatterns exhibit excellent uniformity of submicron sized features within and between arrays, NanoInk's miniaturized and multiplexed assays achieve up to single femtogram/mL assay sensitivity. As a result, 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 <6 [micro]m-wide features in the area occupied by just one spot on a conventional protein microarray, the NanoInk system can pattern as many as 96 sub-arrays on one nanoarray slide. This small scale and exquisite precision enables highly multiplexed protein analysis on a single slide.

Nanopatterning of multiple proteins at subcellular scales also enables the precise control of the cellular microenvironment and functional manipulation of single cells. NanoInk's platform has the ability to precisely pattern features that allow for the co-culture of multiple cell types within microns of each other by binding to sub-cellular extra-cellular matrix proteins.

Nanoscale patterning

At the heart of Nanolnk's ability to miniaturize life science research is its new generation of DPN instrument systems, accessories, assay kits, software and contract services that are suited for proteomics, microstructure functionalization and cell culture work. DPN is the only commercially available technology platform capable of simultaneously depositing 50 nm to 10 pm features of multiple materials with nanoscale accuracy and precision. Using NanoInk's benchtop systems and software, researchers can directly print biomolecules and biocompatible polymers over large pattern areas on substrates like glass slides, plastic, gold and silicon, all under biocompatible conditions.

The NLP 2000 System is a user-friendly and easy-to-operate benchtop instrument with the ability to design and deposit a wide range of patterns on the benchtop. It is ideal for applications that require 1 to 10 pm printing, such as biosensor functionalization and cell nanopatterning studies. Using NanoInk's proprietary MEMs devices and deposition protocols with the NLP 2000 System's printing and automation software and high-resolution optics, users can create custom-patterned substrates in under one hour.

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A second benchtop system, the NanoArray Assay System, is specially designed for nanoscale protein analysis and discovery. Coupling powerful array deposition techniques with the latest generation of fluorescent detection and analysis technologies, the NanoArray Assay System is an affordable desktop unit that can quickly and cost-effectively integrate into any proteomic lab. The platform can create highly reproducible protein arrays over millimeter areas with nanometer precision, and then fluorescently image these nanoarrays with 0.5 pm resolution. When combined with NanoInk's line of fully automatable proteomic assays, the NanoArray Assay System is a complete solution for the detection, identification and quantitation of clinically relevant proteins from a variety of sample types.

The DPN family of systems can simultaneously deposit multiple capture antibodies on a slide to create a minute immunoassay platform. Printed antibody features exhibit highly uniform spot morphology, so multiplexed nanoarray immunoassays are sensitive enough to detect, identify and quantitate clinically relevant, low-abundance proteins from a variety of sample types, for applications such as biomarker analysis, translational medicine and toxicology.

NanoInk assays are SBS-compliant, providing the choice of automated high-throughput proteomic analysis using commercially available lab automation systems or simple benchtop testing using standard immunoassay protocols. Both formats deliver rapid reaction kinetics within miniaturized reagent volumes. Completely configured assay kits are available for simultaneously evaluating a panel of 10 human cytokines. Life science researchers who have difficulty extracting usable protein bio-arker data from limited sample volumes find that Nanolnk's nano-array-based immunoassay format enables them to successfully analyze more samples--faster.

Microstructure functionalization

Because the NanoInk platform can deposit sub-10 pm features with nanoscale precision at addressable locations, it is ideal for functionalizing prefabricated microstructures like biosensors, chemical sensors, micropillars and microelectrodes. Technical hurdles involved in functionalizing sensor arrays with sub-10 micron sensing areas and the inability of traditional functionalization methods to treat multiple sensing areas differently have limited the widespread use of smaller sensing elements. The ability of DPN to directly deposit multiple biomolecules on miniaturized sensing areas as part of an easy and reliable biosensor functionalization methodology serves to overcome these hurdles. As a result, the DPN family of systems can help the scientific community realize the potential of MEMS-based biosensors.

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The ease with which DPN can construct complex patterns of biological materials at sub-cellular scales, attach live single cells to these patterned features and precisely control cell microenvironments makes DPN a powerful cell biology solution.

Micropatterned single cells can be harnessed to probe underlying mechanisms of cell behavior like cell, morphology, adhesion, migration, differentiation, polarization and stem cell lineage. Different cell types micropatterned in close proximity to each other are also ideal for controlled co-culture studies. Until now, co-cultures have proven to be useful for mimicking the in vivo environment and studying effects on stem or progenitor cell function, but the number of experimental variables has made these studies difficult to conduct. Unlike conventional co-culture techniques, the NanoInk platform can spatially manipulate multiple cell types at single cell levels on a substrate, allowing the user to better control co-culture experimental variables.

To pattern co-cultured cells, DPN is first used to deposit extracellular matrix proteins on a substrate. Cells are plated and preferentially bind to these ECM domains. NanoInk has conducted proof-of-concept studies involving 3T3 fibroblasts and C2C12 myoblasts bound respectively to fibronectin and laininin features to validate this single cell co-culture concept. Other potential techniques for DPN patterning of co-cultures include using printed cell binding ligands to subsequently bind cells that express the corre-sponding receptors, or depositing single-strand DNA to then bind cells with complementary DNA on their cell surface.

The NanoInk platform can control the shape and number of bound cells by varying the underlying pattern geometry and total patterned area of deposited extracellular matrix proteins. This is an important feature because cluster size and cell morphology are known to play crucial roles in the function of many types of cells.

Researchers can also deliver compounds of interest to cells by harnessing the properties of hydrogels using the NanoInk platform's ability to pattern sub-10 micron polymer materials at defined locations in a controlled, consistent manner. Scientists can expose individual cells to compounds of interest loaded in hydrogels and then study the biological effect of these materials at the single cell level. DPN-generated hydrogels show promise not only for cell morphology studies but also for exploring cell-substrate and protein-cell interactions, as well as tissue engineering and in vitro cell culture studies.

AT A GLANCE

* Dip Pen Nanolithography (DPN) can rapidly create micro and nanoscale patterns.

* The NLP 2000 System can design and deposit a range of patterns on a benchtop.

* Micropatterned single cells can be harnessed to probe underlying mechanisms of cell behavior.

CONTACT

For more information, please contact NanoInk at 847-679-6266, or visit www.nanoink.net.

by Tom Warwick, Nanolnk, Inc., Chicago, III.
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Title Annotation:Feature
Author:Marwick, Tom
Publication:Laboratory Equipment
Date:Aug 1, 2012
Words:1410
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