( By Linda E. Cammi...)New Tools for Making and Processing Protein Microarrays.

By Linda E. Cammish, Ph.D., NextGen Sciences, Ltd. and Mark N. Bobrow, Ph.D., PerkinElmer Life Sciences, Inc.

Introduction

The use of DNA microarrays in scientific research and drug discovery has become very widespread. However, since it is proteins and not genes that are the vast majority of the true drug targets, there has been a fast growing interest in the use of functional protein microarrays, sometimes referred to as protein biochips. Unlike DNA molecules, functional proteins are not as easy to attach to a biochip substrate. Proteins also exhibit a wider range of physical characteristics, these being determined by the charged or non-charged functional side groups, regions of hydrophobicity or hydrophilicity and the manner in which the protein is folded via covalent or non-covalent interactions.

The production of protein microarrays is therefore fraught with many more problems than those encountered in the use of DNA microarrays. In addition, the use of protein microarrays requires that the assays performed be highly optimized due to the fact that there are many more variables to deal with including different pH conditions, buffer compositions, detergent types and concentrations, blocking reagents to prevent non-specific interactions and also the way in which the proteins are attached to substrates. All of these parameters have a significant impact on the affinity and specificity of protein-protein or protein-ligand interactions.

Despite these challenges, multiple protein assays that are performed in parallel, in miniature and in the format of a protein array hold great potential for target discovery and validation. To support this development process, there is a growing need to be able to automate these assays to improve sensitivity and reproducibility, and to make the optimization of each assay quicker and easier for the researcher to perform. To achieve this, a protein microarray processor must be a highly flexible system, much more flexible than that required for processing DNA arrays, which is a technology that has matured very rapidly over the last 5 years.

PerkinElmer Life Sciences has recently introduced the ProteinArray Workstation (Figure 1), the first fully automated system for processing protein microarrays. This coincides with the launch of the companys new SpotArray and ScanArray Express systems for the respective printing and analyzing of both DNA and protein microarrays. Combining these three new systems with the novel 3-dimensional HydroGel substrate and TSA (Tyramide Signal Amplification) chemistries enables PerkinElmer Life Sciences to offer researchers an unprecedented and complete suite of technologies specifically designed for protein microarray applications.

The ProteinArray Workstation was developed as part of a collaborative development agreement between NextGen Sciences and PerkinElmer Life Sciences. Based on NextGen Sciences novel microfluidics technology, the ProteinArray Workstation is a highly flexible, fully automated system for processing multiple protein microarrays. It has a series of unique features and has been specifically designed for processing protein microarrays based on microscope slide formats, including the HydroGel substrate. The ProteinArray Workstation has been used to automate and miniaturize many of the applications for protein microarray technologies that have been described in the literature, including protein expression analysis, protein-protein interaction studies and antibody profiling. It is suitable for use with any application where the high throughput analysis of multiple proteins is required.

Key Advantages

The advantages of the ProteinArray Workstation over existing technologies include its ability to analyze multiple proteins in parallel with very short processing times and, more importantly, to achieve this in a very low volume (75 μL) reaction chamber. The ability to perform multiple, miniaturized assays is particularly advantageous for research that requires expensive reagents, or where only a limited amount of clinical sample or biopsy material is available for analysis.

Since temperature control of any protein assay is essential for reproducibility between a series of experiments, the ProteinArray Workstation has been designed to ensure complete uniformity of temperature to within 0.5C, not only across the protein array during the assay but also between reaction chambers. The samples are stored in a thermally controlled block prior to being introduced into the reaction chamber, so no off-line heating or cooling of the sample is required. There is also precise, stable temperature management of the protein microarray from 4 to 45C, enabling flexibility in assay design.

A key aspect of the ProteinArray Workstation is that it is fully automated. Once reagents and samples are loaded onto the instrument, no further manual intervention is required. Even the introduction of up to 2 low volume (75 μL) samples per protein microarray into the biochip reaction chamber, is performed automatically. The unique ability of the system to automatically introduce two low volume samples enables the system to be used to automate applications such as sandwich assays, where a second sample comprising an antibody that is unique to the assay and may be highly valuable is required. The result is a significant reduction in hands-on time and in the total processing duration, releasing the operator for other activities. Additionally, multiple protein microarrays can be processed in parallel, enabling high throughput, predictable and reproducible performance of assays without scheduling difficulties.

Another unique aspect of the system is its ability to perform on-line mixing of buffers via the use of multiple, dedicated, low pulsation, peristaltic pumps. This enables selective blending between three bulk liquid channels, allowing use of stock buffer concentrates that can be mixed to user-specified concentrations. This removes the need to make up different buffers in advance when developing assays, allows for rapid method development, and supports the facility to perform gradient washes over the protein microarray surface, thus allowing unique flexibility in assay design. The pumps can be programmed with user definable flow rates (50 μL to 400 μL per minute, equivalent to 1 to 8 volume changes per minute across the biochip) enabling assays incorporating high affinity antibodies to be processed with higher flow rates and shortened assay times (Figure 2).

The TSA Module on the ProteinArray Workstation enables automation of a series of novel protocols that PerkinElmer Life Sciences has developed to boost the sensitivity of protein array-based assays1,2. These multi-step protocols provide for signal amplification through the use of tyramide derivatives as a substrate for horse radish peroxidase (HRP). This catalyzes a reaction to form activated tyramides which rapidly bind to multiple tyrosine residues in any proteins that are immediately adjacent to the HRP conjugate. Using a series of different tyramide derivatives and antibody detection systems, amplification can be achieved with virtually any protein assay. The amplification provides for an increase in sensitivity of up to 100-fold providing researchers with the ability to detect low abundance proteins in complex mixtures, including clinical samples.

When performed manually, the multi-step TSA assays can be very time consuming and laborious. To automate this process the ProteinArray Workstation includes the TSA Module. This automates the delivery of up to 6 reagents, including the TSA chemistries, to each of the protein arrays being processed. The module can also be used for delivery of valuable reagents such as antibodies, or streptavidin, which are labelled with dye or enzyme.

Description of the ProteinArray Workstation

A ProteinArray Workstation is comprised of:

(1) a Reagent Delivery System and

(2) up to 4 ProteinArray Processors.

The Reagent Delivery System supplies the 6 bulk buffers to the ProteinArray Processor, or Processors, as well as pressure and vacuum lines that control the system. All of the reagent reservoirs are contained within an enclosure enabling safe storage of buffers and reducing dust contamination.

The ProteinArray Processors can be stacked to minimize the system footprint and save laboratory bench space. Each ProteinArray Processor can be operated independently and can process up to 12 protein microarrays in parallel. As each successive ProteinArray Processor is incorporated the total protein microarray capacity of the entire system increases from 12 to 24, to 36 or to 48 microarrays, with each Processor remaining independently controllable.

Once one Processor has been installed, the system can be easily upgraded by adding further Processors, as the researchers needs increase. This enables the system to be used by a small or large research laboratory, by single or multi-users, or even in a core facility where high throughput capability in a multi-user environment is required. Since the Processors are independently controlled, different assay methods can be processed concurrently on each Processor, enabling high throughput, flexible operation and fast method development.

Core to the ProteinArray Processor is the unique design of the microfluidics and sample injection system. Each ProteinArray Processing Cell is comprised of a stack of components with micro channels and valves that are firmly clamped into position and are not normally disassembled by the operator (Figure 3).

The Processing Cell includes a base block, which provides the route for fluid channels, creation of micro-valves and 2 low-volume sample storage chambers. On top of this base block is a membrane diaphragm, a further micro channel block to create fluid paths, another membrane diaphragm and finally the reaction chamber block which is temperature controlled. This provides for fluid input/output to the reaction chamber and forms the base of the reaction chamber itself. The reaction chamber block also contains the pipette tip holding mechanisms and ports by which the low-volume samples are added to the sample storage chamber.

The reaction chamber is created between the surfaces of the reaction chamber block and the protein microarray. These are separated by a very narrow shim or gasket, the thickness of which determines the reaction volume over the microarray surface and can be varied to accommodate either the planar glass biochips or the 3-dimensional surface of the HydroGel coated slides.

To simplify loading protein microarrays into the ProteinArray Workstation, the biochip is placed into a protein microarray carrier, which also incorporates the shim or gasket. The protein microarray is firmly clamped into position using a single action mechanism providing thorough sealing around the extreme edge of the biochip substrate and giving an active area of 21 mm x 60 mm (Figure 4).

The low-volume sample is loaded into the sample storage chamber by drawing 75 μL of sample into a pipette tip (Eppendorf, Gilson or similar). The pipette tip, complete with 75 uL of sample, is positioned in the pipette tip holding mechanism. The sample is then automatically drawn into the sample storage chamber as part of the protocol at the start of a method. The sample remains in the sample storage chamber during automated processing until the point at which it is needed in the assay, when it is then automatically delivered onto the surface of the protein microarray in the reaction chamber via the micro-fluidics system.

The ProteinArray Workstation can be programmed to automatically agitate the sample back and forth across the surface of the protein microarray to provide an even concentration of the analyte throughout the entire sample volume. After incubation on the protein microarray, the sample is removed and the protein microarray rinsed with wash buffers prior to the next step in the assay.

The ProteinArray Workstation is computer controlled to provide for fully automated processing. The user interface is similar to Microsoft Office productivity software. Methods for each assay can be created, edited and stored by a drag-and-drop operation, making it very easy to learn and use. To further simplify operation, the software is also provided with pre-defined, optimized, template protocols that the user can copy and edit to rapidly create new methods to run.

Before running a method, the ProteinArray Workstation performs a validation step to check that all the steps that have been programmed are meaningful and possible to perform. The system also keeps a method status log of all the methods running on all ProteinArray Processors, providing users with reassurance that assays were performed exactly as programmed and with a record of any user interventions.

Live progress monitoring is included in the software, which displays an overview of the entire method in a Gantt chart format. This is updated in real-time and enables operators to observe the length and status of any assay step as it is performed, again providing operator reassurance. The ProteinArray Workstation also allows for the inclusion of a barcode reader for protein microarray/ sample/reagent/instrument identification, confirmation and input into log files enabling easy tracking via a Laboratory Information Management System (LIMS).

Summary

The ProteinArray Workstation can be used to fully automate processing of multiple protein microarrays. The system is both flexible and reproducible.

Processing protein microarrays is only one element involved in the workflow of utilizing protein microarrays. Before the biochip can be used, proteins need to be arrayed onto the biochip substrate. PerkinElmer Life Sciences has a whole range of arraying systems, including the BioChip Arrayer, the SpotArray 24, the SpotArray 72 and the SpotArray Enterprise for a range of different user needs from research and development, to high throughput and production. The BioChip Arrayer and the SpotArray Enterprise feature PiezoTipnology, a highly precise form of non-contact ink-jet technology that does not heat samples during dispensing. The SpotArray 24 and 72 are split-pin based contact printers allowing for rapid creation of high-density arrays. After processing, protein microarrays need to be scanned to enable detection. Again PerkinElmer Life Sciences offers a range of scanning systems including the ScanArray Lite , the ScanArray Express and the ScanArray Express HT to meet the range of different user needs.

With the introduction of the ProteinArray Workstation, PerkinElmer Life Sciences is in the unique position to be able to offer a complete suite of products to provide users with a total solution for protein microarrays. This range includes:

Protein Microarray Substrate HydroGel coated slides

Protein Microarray Printing PiezoTipnology

Protein Microarray Processing ProteinArray Workstation

Protein Microarray Labeling and Amplification Cyanine dyes and TSA

Protein Microarray Scanning ScanArray Express

Protein Microarray Data Analysis QuantArray

Acknowledgements

The authors thank Ian Taylor, Ph.D. (Director of Proteomics, PerkinElmer Life Sciences, 204 Cambridge Science Park, Milton Road, Cambridge, CB4 0GZ, UK) and Kevin Auton, Ph.D. (NextGen Sciences) for their contributions and comments in writing this article.

References

1. M. N. Bobrow and G.J. Litt: Method for detection or quantitation of an analyte using an analyte dependent enzyme activation system. US Patent 5,196,306.

2. M. N. Bobrow and G.J. Litt: Catalyzed Reporter Deposition. US Patent 5,731,158.

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