A plate of chips

Overview

DNA and protein microarrays printed on glass or membrane-coated microscope slides are widely used in scientific research today. This approach has provided researchers with the opportunity to investigate thousands of gene sequences or proteins in a single experiment. However, there is a growing demand for even higher throughput microarray assays.

Genomic Solutions, the market leader in DNA and protein microarraying, now offers the capability to multiplex array experiments by printing microarrays into the wells of microtiter plate wells. This technology is available on both the MicroGrid II and the OmniGrid Accent microarrayers: the former holds up to 16 target microplates, while the latter is capable of holding up to 6 target microplates. Arrays can be printed into 96- or 384-well plates, and in a circular or square layout, to maximize use of the available area in the wells.

Hardware and software upgrades are available for both instruments, allowing users currently printing on slides to easily convert to plate arraying. In addition, it is very easy to switch between the two applications; a useful feature for core laboratories with multiple customers performing different types of assays.

A typical experiment might involve printing the same set of samples into each well of a microtiter plate and then hybridizing each array with a different test and/or control sample. In this way you can perform 96 different assays at the same time on a single substrate. Since each well can hold over 1000 sample spots, this represents over 1.6 million individual hybridization assays if the Microgrid was used to its full capacity. This obviously represents a significant increase in throughput.

Since sample stability might be an issue when performing longer print runs, both microarrayers can maintain a cooled environment for the source plates. This is especially important for protein printing, which is rapidly gaining popularity in the research community. Protein samples are often printed onto membrane-coated surfaces, and both of the arrayers mentioned have customizable parameters designed to optimize this process and prevent damage to the often delicate surfaces.

Another advantage of printing into microplates is that there are many instruments readily available to automate processing of these 8 x 12cm substrates. This makes the technology more attractive to high throughput assays -- for example testing a battery of unknown compounds for reactivity against a known library of substrates. We envision that such a multiplexed approach will enable researchers to drive the advancement of microarray production into new application areas such as micro ELISA assays, diagnostic arrays, high throughput protein interaction studies and chemical or other small molecule screening.

The BioRobotics MicroGrid II has the capacity to hold 16 microplates while the GeneMachines OmniGrid Accent is capable of holding 6 microplates. In both cases this enables a researcher to improve assay production by up to a factor of fifty, compared to current microarray methods.

In the following tutorial, we will list some of the common challenges for plate arraying and protein arraying and our solutions to these challenges.

Plate Arraying

1. Plate arrays—the challenges:

• Production of high density multiplexed arrays
• Increasing throughput to allow walk-away automation
• Depositing samples onto delicate surfaces
• Avoiding cross contamination
• Maintaining sample stability
• Minimize dust contamination within arrays
  

2. Plate arrays—details on our solutions:

High density multiplexed arrays
Adapter plates are available for both instruments enabling the user to print arrays into individual wells of microplates rather than onto slides. The GeneMachines OmniGrid Accent can array into 6 destination plates whereas the BioRobotics MicroGrid II has three times greater capacity, holding 16 microplates. The Genomic Solutions microarray platform has been designed with flexibility as a priority and can accommodate many plate definitions for this application. A library of definitions for most commonly available microplates is available in the software allowing simple set up of arrays into the users’ choice of plates.

In house research has shown it is possible to array and accurately quantitate at least 1000 DNA or protein spots in a single well of a 96 well microplate or 450 features in each well of a 384 well microplate. Arrays can be printed in either a circular or square format to ensure maximum use of well space.

Genomic Solutions provides flexibility in plate configuration enabling researchers to array from 96 wells into 384 wells and vice versa as well as between plates of the same format.

These high density arrays are produced using contact printing with Telechem 946 pins or MicroSpot 2500 or 10K split pins*. These split-pin designs load up to 250nl of sample into the pin by capillary action, covering the end of the pin tip with a thin layer and allow printing by simple surface tension and adhesion. This benefits the researcher by enabling samples to be deposited onto delicate surfaces with minimal contact between pin and substrate.

Due to the viscous nature of proteins researchers may choose to move away from split-pin technology for the plate arraying of proteins. Genomic Solutions can offer these scientists the option of proprietary MicroSpot solid pin technology.

Increasing throughput with walk-away automation

The GeneMachines OmniGrid Accent can hold 3 source plates and 6 target microplates. The BioRobotics MicroGrid II has almost three times the target plate capacity and can hold 24 source plates in a cooled environment. These configurations enable a researcher to improve assay production by up to a factor of fifty, compared to standard slide production methods. Both instruments are compatible with 96 and 384 well microplates.

To enhance walk-away usability further Genomic Solutions has also developed microarray software for plate-based applications. Both instruments have flexible software offering a fully customizable array design feature a simple wizard guides the operator through target plate selection, pin tool configuration and source options. The user is then taken through array design and layout as well as pin washing options. After a summary of the parameters selected the plate-arraying run will begin.

An essential sample tracking function within the software ensures traceability from source plate well to individual spot location within each microplate well by the production of a .gal file.

Depositing samples onto delicate surfaces

Control of spotting speed using Genomic Solutions proprietary Soft Touch feature allows precise and gentle printing onto the surface of the well, particularly useful when arraying onto a coated surface. It improves spot morphology and also reduces pre-spotting enabling higher throughput and ensuring valuable sample is not wasted.

In more detail, when you are moving your array printhead around to spot your pins onto your array substrates, you move it at a particular speed and normally the speed is very fast. Soft Touch allows you to quickly decelerate the pin to a very slow speed just before the point of contact. This gives you much better spot definition and morphology by doing this quick deceleration process just before you touch. Once you have finished the touch, it re-accelerates back to the original speed.

A screen shot from BioBioRobotics MicroGrid II Plate array software

Avoiding cross contamination

Thorough pin cleaning is extremely important for successful arraying, especially for protein applications; therefore Genomic Solutions offers pin sonication in addition to multiple wash stations.

Maintaining sample stability

Genomic Solutions arrayers can include source plate cooling. This is especially important for helping to ensure that proteins remain stable prior to transfer to the substrate surface. Both the BioRobotics MicroGrid II and the GeneMachines OmniGrid Accent have humidification as standard to prevent sample evaporation.

Reduce dust contamination within arrays

Both the BioRobotics MicroGrid II and the GeneMachines OmniGrid Accent use HEPAfiltration to minimize dust present within the system, ensuring the printing of clean, high quality arrays.

Protein Arraying

3. Protein arrays—challenges:

Protein microarrays offer the ability to measure protein expression and antibody binding in a high-throughput and miniaturized manner. Over the years, Genomic Solutions has developed instruments to enable automated workflow process of DNA microarray production, hybridization and data analysis. The ability to use these instruments for protein microarrays greatly increases their functionality and streamlines the process of generating data. Unfortunately, there has been a lack of information on the processes involved in using DNA microarray equipment for the protein microarray workflow. Protein microarrays have some unique features that may make DNA microarray instrumentation difficult to transition. Here are some of the common challenges relating to protein arraying:

• Hard to set up protein array parameters
• Concerns on carry-over contamination
• Concerns over protein-protein binding
  

4. Protein arrays—our solutions to the challenges:

Here are some of our solutions to overcome these challenges:

Optimization of Protein Array Parameters

A major problem with the optimization of protein array printing is the time and cost associated with the set-up and analysis of protein-protein interactions. Optimization using fluorescent dye eliminates the use of costly experimental samples in order to evaluate the optimization process. We recommend using some type of fluorescent substance (e.g. red food dye resuspended in 1X PBS) to allow visual inspection of spot deposition. By scanning slides printed with red food dye in the Cy3 channel it is possible to get a high resolution image of the spots to use for optimizing printing conditions.

Minimizing Carry-over Contamination

Due to the composition and differing viscosity of various spotting solutions used for protein arrays, minimizing the amount of carry-over between spots is a critical step for successful protein arraying. The Genomic Solutions Omnigrid and Microgrid microarraying systems allow for complete control over the wash steps and cycles.

In order to establish a wash protocol that did not have carry-over, each dip into the dye sample was spotted 6 times onto the slides followed by the wash cycle, dipped into 1X PBS without dye and printed for an additional 6 spots. This was repeated three times. By varying the number of cycles through the sonication, wash, and dry cycles it is possible to develop a wash protocol that does not have any carry-over between samples.

The Omnigrid system has the advantage of an on board sonication system to increase the effectiveness of the wash cycles. The Microgrid system has two recirculating wash baths and uses the flood and flush method of cleaning the pins. Additionally, the two recirculating wash baths on the Microgrid can be used as stationary baths, which can be filled with various cleaning agents such as ethanol. Although the 2 systems have different wash setups, wash methods on both systems were shown to be equally effective for cleaning pins.

Due to variations in printing solutions and concentration of proteins, sample specific wash protocols will need to be determined empirically. By varying the times and number of cycles through the wash stations, it is possible to print proteins on nitrocellulose without any visible carry-over.

Protein-Protein Binding

When proper printing and wash protocols have been established, the next steps are printing followed by addition of the solution-phase binder. Protein arrays may have complex binding protocols involving multiple blocking and wash steps. The Genomic Solutions Hybstation is a convenient way to automate complex solution-phase binding procedures.

Standard protein solution-phase binding protocol can be written using the Methods Editor software program of the Hybstation. Blocking, solution-phase binding, and wash steps can be included in the protocol and antibody binding may be carried out overnight. The slides were then allowed to air dry prior to scanning. This basic program can be modified and adjusted easily with the Methods Editor software according to the individual researcher needs.

SUMMARY

In conclusion, there are several challenges to the production of plate arrays. Genomic Solutions can now offer high quality plate arraying as an option or upgrade to the existing BioRobotics MicroGrid and the GeneMachines OmniGrid Accent. This technology is part of a suite of products from Genomic Solutions designed to assist the researcher in meeting the challenges of achieving a multiplexed approach to DNA and protein microarraying. With this technology advance, plate and protein arraying may finally become a reality to serve the need of more diagnostic applications.

For a demonstration of this new technology or advice on you current microarray applications, please contact us at: arraydesign@genomicsolutions.com

*MicroSpot split pins not available in USA or Canada