www.perkinelmer.com/proteomics

Protein Microarrays

Drug Discovery Clinical Screening

ProteinArray Workstation™

New Tools for Making andProcessing Protein Microarrays

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

Research

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IntroductionThe use of DNA microarrays in scientific research anddrug discovery has become very widespread. However,since it is proteins and not genes that are the vast majorityof the true drug targets, there has been a fast growing inter-est in the use of functional protein microarrays, sometimesreferred to as protein biochips. Unlike DNA molecules,functional proteins are not as easy to attach to a biochipsubstrate. Proteins also exhibit a wider range of physicalcharacteristics, these being determined by the charged ornon-charged functional side groups, regions of hydropho-bicity or hydrophilicity and the manner in which theprotein is folded via covalent or non-covalent interactions.

The production of protein microarrays is therefore fraughtwith many more problems than those encountered in theuse of DNA microarrays. In addition, the use of proteinmicroarrays requires that the assays performed be highlyoptimized due to the fact that there are many more vari-ables to deal with including different pH conditions, buffercompositions, detergent types and concentrations, block-ing reagents to prevent non-specific interactions and alsothe way in which the proteins are attached to substrates.All of these parameters have a significant impact on theaffinity and specificity of protein-protein or protein-ligandinteractions.

Despite these challenges, multiple protein assays that areperformed in parallel, in miniature and in the format of aprotein array hold great potential for target discovery andvalidation. To support this development process, there isa growing need to be able to automate these assays toimprove sensitivity and reproducibility, and to make theoptimization of each assay quicker and easier for theresearcher to perform. To achieve this, a protein microarrayprocessor must be a highly flexible system, much moreflexible than that required for processing DNA arrays,which is a technology that has matured very rapidly overthe last 5 years.

PerkinElmer Life Sciences has recently introduced theProteinArray Workstation (Figure 1), the first fullyautomated system for processing protein microarrays.This coincides with the launch of the company’s newSpotArray™ and ScanArray™ Express systems for therespective printing and analyzing of both DNA and proteinmicroarrays. Combining these three new systems with thenovel 3-dimensional HydroGel™ substrate and TSA™

(Tyramide Signal Amplification) chemistries enablesPerkinElmer Life Sciences to offer researchers an unprece-dented and complete suite of technologies specificallydesigned for protein microarray applications.

The ProteinArray Workstation was developed as part of acollaborative development agreement between NextGenSciences and PerkinElmer Life Sciences. Based onNextGen Sciences’ novel microfluidics technology, theProteinArray Workstation is a highly flexible, fully auto-mated system for processing multiple protein microarrays.It has a series of unique features and has been specificallydesigned for processing protein microarrays based onmicroscope slide formats, including the HydroGel sub-strate. The ProteinArray Workstation has been used toautomate and miniaturize many of the applications forprotein microarray technologies that have been describedin the literature, including protein expression analysis,protein-protein interaction studies and antibody profiling.It is suitable for use with any application where the highthroughput analysis of multiple proteins is required.

Figure 1. The ProteinArray Workstation

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The advantages of the ProteinArray Workstation overexisting technologies include its ability to analyze multi-ple proteins in parallel with very short processing timesand, more importantly, to achieve this in a very lowvolume (75 µL) reaction chamber. The ability to performmultiple, miniaturized assays is particularly advantageousfor research that requires expensive reagents, or whereonly a limited amount of clinical sample or biopsy materi-al is available for analysis.

Since temperature control of any protein assay is essentialfor reproducibility between a series of experiments, theProteinArray Workstation has been designed to ensurecomplete uniformity of temperature to within 0.5°C, notonly across the protein array during the assay but alsobetween reaction chambers. The samples are stored in athermally controlled block prior to being introduced intothe reaction chamber, so no off-line heating or cooling ofthe sample is required. There is also precise, stable tem-perature management of the protein microarray from 4 to45°C, enabling flexibility in assay design.

A key aspect of the ProteinArray Workstation is that it isfully automated. Once reagents and samples are loadedonto the instrument, no further manual intervention isrequired. Even the introduction of up to 2 low volume (75µL) samples per protein microarray into the biochip reac-tion chamber, is performed automatically. The uniqueability of the system to automatically introduce two lowvolume samples enables the system to be used to auto-mate applications such as sandwich assays, where asecond sample comprising an antibody that is unique tothe assay and may be highly valuable is required. Theresult is a significant reduction in hands-on time and inthe total processing duration, releasing the operator forother activities. Additionally, multiple protein microarrayscan be processed in parallel, enabling high throughput,predictable and reproducible performance of assayswithout scheduling difficulties.

Another unique aspect of the system is its ability toperform on-line mixing of buffers via the use of multiple,dedicated, low pulsation, peristaltic pumps. This enablesselective blending between three bulk liquid channels,allowing use of stock buffer concentrates that can bemixed to user-specified concentrations. This removes theneed to make up different buffers in advance when devel-oping assays, allows for rapid method development, andsupports the facility to perform gradient washes over theprotein microarray surface, thus allowing unique flexibili-ty in assay design. The pumps can be programmed withuser 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 affinityantibodies to be processed with higher flow rates andshortened assay times (Figure 2).

The TSA Module on the ProteinArray Workstation enablesautomation of a series of novel protocols that PerkinElmerLife Sciences has developed to boost the sensitivity ofprotein array-based assays1,2. These multi-step protocolsprovide for signal amplification through the use of tyra-mide derivatives as a substrate for horse radish peroxidase(HRP). This catalyzes a reaction to form activated tyra-mides which rapidly bind to multiple tyrosine residues inany proteins that are immediately adjacent to the HRPconjugate. Using a series of different tyramide derivativesand antibody detection systems, amplification can beachieved with virtually any protein assay. The amplifica-tion provides for an increase in sensitivity of up to100-fold providing researchers with the ability to detectlow abundance proteins in complex mixtures, includingclinical samples.

When performed manually, the multi-step TSA assays canbe very time consuming and laborious. To automate thisprocess the ProteinArray Workstation includes the TSAModule. This automates the delivery of up to 6 reagents,including the TSA chemistries, to each of the proteinarrays being processed. The module can also be used fordelivery of valuable reagents such as antibodies, or strep-tavidin, which are labelled with dye or enzyme.

Key Advantages

Figure 2. Organization of buffers, high-value reagents and samples in the ProteinArray Workstation.

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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 buffersto the ProteinArray Processor, or Processors, as well aspressure and vacuum lines that control the system. All ofthe reagent reservoirs are contained within an enclosureenabling safe storage of buffers and reducing dustcontamination.

The ProteinArray Processors can be stacked to minimizethe system footprint and save laboratory bench space. EachProteinArray Processor can be operated independently andcan process up to 12 protein microarrays in parallel. Aseach successive ProteinArray Processor is incorporated thetotal protein microarray capacity of the entire systemincreases from 12 to 24, to 36 or to 48 microarrays, witheach Processor remaining independently controllable.

Once one Processor has been installed, the system canbe easily upgraded by adding further Processors, as theresearchers’ needs increase. This enables the system to beused by a small or large research laboratory, by single ormulti-users, or even in a core facility where high through-put capability in a multi-user environment is required.Since the Processors are independently controlled, differ-ent assay methods can be processed concurrently on eachProcessor, enabling high throughput, flexible operationand fast method development.

Core to the ProteinArray Processor is the unique designof the microfluidics and sample injection system. EachProteinArray Processing Cell is comprised of a stack ofcomponents with micro channels and valves that arefirmly clamped into position and are not normally disas-sembled by the operator (Figure 3).

The Processing Cell includes a base block, which providesthe route for fluid channels, creation of micro-valves and2 low-volume sample storage chambers. On top of thisbase block is a membrane diaphragm, a further microchannel block to create fluid paths, another membranediaphragm and finally the reaction chamber block whichis temperature controlled. This provides for fluidinput/output to the reaction chamber and forms the baseof the reaction chamber itself. The reaction chamber blockalso contains the pipette tip holding mechanisms andports by which the low-volume samples are added to thesample storage chamber.

The reaction chamber is created between the surfaces ofthe reaction chamber block and the protein microarray.These are separated by a very narrow shim or gasket, thethickness of which determines the reaction volume overthe microarray surface and can be varied to accommodateeither the planar glass biochips or the 3-dimensionalsurface of the HydroGel™ coated slides.

Figure 3. Processing Cell with new microfluidics system. The micro channels and micro valves incorporated into the ProteinArray Workstation are created through a multi-layered stack.

Description of the ProteinArray Workstation

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To simplify loading protein microarrays into theProteinArray Workstation, the biochip is placed into aprotein microarray carrier, which also incorporates theshim or gasket. The protein microarray is firmly clampedinto position using a single action mechanism providingthorough sealing around the extreme edge of the biochipsubstrate and giving an active area of 21 mm x 60 mm(Figure 4).

The low-volume sample is loaded into the sample storagechamber by drawing 75 µL of sample into a pipette tip(Eppendorf, Gilson or similar). The pipette tip, completewith 75 uL of sample, is positioned in the pipette tipholding mechanism. The sample is then automaticallydrawn into the sample storage chamber as part of theprotocol at the start of a method. The sample remains inthe sample storage chamber during automated processinguntil the point at which it is needed in the assay, whenit is then automatically delivered onto the surface of theprotein microarray in the reaction chamber via themicro-fluidics system.

The ProteinArray Workstation can be programmed to auto-matically agitate the sample back and forth across thesurface of the protein microarray to provide an evenconcentration of the analyte throughout the entire samplevolume. After incubation on the protein microarray, thesample is removed and the protein microarray rinsed withwash buffers prior to the next step in the assay.

The ProteinArray Workstation is computer controlled toprovide for fully automated processing. The user interfaceis similar to Microsoft Office® productivity software.Methods for each assay can be created, edited and storedby a “drag-and-drop” operation, making it very easy tolearn and use. To further simplify operation, the softwareis also provided with pre-defined, optimized, templateprotocols that the user can copy and edit to rapidly createnew methods to run.

Before running a method, the ProteinArray Workstationperforms a validation step to check that all the steps thathave been programmed are meaningful and possible toperform. The system also keeps a method status log of allthe methods running on all ProteinArray Processors,providing users with reassurance that assays wereperformed exactly as programmed and with a record ofany 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 andenables operators to observe the length and status of anyassay step as it is performed, again providing operatorreassurance. The ProteinArray Workstation also allows forthe inclusion of a barcode reader for protein microarray/sample/reagent/instrument identification, confirmationand input into log files enabling easy tracking via aLaboratory Information Management System (LIMS).

Figure 4. Protein microarray, shim and slide carrier located in position face-down on the reaction chamber.

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SummaryThe ProteinArray Workstation can be used to fully automate processing of multiple protein microarrays. Thesystem 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 Scienceshas a whole range of arraying systems, including the BioChip Arrayer, the SpotArray 24, the SpotArray 72 andthe SpotArray Enterprise for a range of different user needs from research and development, to high throughputand production. The BioChip Arrayer and the SpotArray Enterprise feature PiezoTipnology™, a highly preciseform of non-contact ink-jet technology that does not heat samples during dispensing. The SpotArray 24 and 72are split-pin based contact printers allowing for rapid creation of high-density arrays. After processing, proteinmicroarrays need to be scanned to enable detection. Again PerkinElmer Life Sciences offers a range of scanningsystems including the ScanArray Lite, the ScanArray Express and the ScanArray Express HT to meet the rangeof different user needs.

With the introduction of the ProteinArray Workstation, PerkinElmer Life Sciences is in the unique position to beable to offer a complete suite of products to provide users with a total solution for protein microarrays. Thisrange 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®

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

References1. 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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