Thursday, September 13, 2007

Microarray manufacturing to perfection

Want to make a perfect microarray? You need 5 things.

1. 946 Micro Spotting Device (US Patent 6,101,946)

This unique device has the capability to spot picoliter volumes of liquids, up to 48 different samples at a time, over hundreds of substrates with only 250 nanoliters of starting volume. The device is incredibly simple, yet unbelievably flexible. If a high number of different biomolecules need to be spotted, a full set of pins can be used in the holder. If a low number of biomolecules need to be spotted, then only 1 pin can be used...or anything in between. Pins float individually in the holder or printhead as it is called, to make adding or removing pins only take a few seconds. This also make maintenance on the tool very easy, since no special tooling or tech visits are required to replace pins in the tool. Spots on microarrays need to be in good proximity to each other for optimal binding kinetics and efficient reaction volumes. The key to the spotting technology is the defined and exterior uptake channel and flat tip (apex of the tip horizontally level to the centerline of the shaft of the pin). These characteristics empower incredibly reliable and simple capillary spotting along with the ability to easily wash the tips and load new samples quickly. This makes the microarraying process go much faster than ink jetting mechanisms that are difficult to clean and are limited in the number of delivery mechanisms. The 946 device's ability to spot multiple samples, multiple times, over multiple substrates, with one low volume loading of sample as made the tool the most widely used microarray product in the world. With the mechanical tolerances of each delivery mechanism being within two microns, the tool has excellent reproducibility and performance. A wide variety of pin tip sizes are available to make different spot sizes, thus microarrays can be made for both high and low resolution detection instruments..

2. NanoPrint Microarrayer

The most important characteristics of a microarray robot is it's accuracy and repeatability and the efficiency of the wash/dry station for the spotting mechanism. Other important things to look for are easy of use and a powerful graphical user interface. The NanoPrint microarrayer exceeds these minimum requirements. The NanoPrint microarraying robot get's its name because it's movements are measured in nanometers and when equipped with a 946 or Stealth Micro Spotting Device, delivers sub nanoliter volumes to a wide variety of solid surfaces including but not limited to glass, membranes and plastics. Since the delivery mechanism of the 946 spotting device is capillary action, accurate movements in the Z axis are most critical to get best performance. The Z axis resolution of the NanoPrint is 250 nanometers (4,000 steps per millimeter), by far the most accurate robot available in the industry. The optical encoders on the system measure and confirm each and every movement the system makes in any given microarray manufacturing run.

3. Microarray Slides Substrates

Microarray substrates take on different forms, as mentioned earlier in this blog, surfaces are typically glass (most commonly), membrane or plastic. In order for the same amount of biomolecule to be immobilized at each microarray location on the surface, the substrate must have a perfectly even distribution capture reactive groups across the entire printing surface. In other words, it must be completely homogeneous in the printed area. A microarray capable of generating quantitative data has the same amount of sample at each array location on the printing substrate. If a dilution series is being printed, the dilution of the biomolecule bound to the surface must match the dilution of the unprinted material. If the substrate is not homogeneous, meaning that it does not have the same amount of reactive groups at each array location on the substrate, then different amounts of sample will attach to the surface and any hope of generating quanitative data will be lost. The glass surface we make here at the TeleChem ArrayIt division are polished so that the topography of the glass does not deviate more than 50 angstoms. This assures that surface chemistry is homogeneous and that results can be quantitative. Recently SuperEpoxy 2 surface chemistry is being used in protein, peptide and DNA microarray applications...making it the most versatile microarray surface chemistry available in industry. The surface has even been used to immobilize serum (click here for paper). The surface has a high binding capacity and the surface is easy to block non-specific binding to keep background low. Microarray detection is always a battle to see more signal and less noise....the lower the background the better.

4. Sample Preparation

Sample preparation is often the most overlooked variable in microarray manufacturing. Problems often associated with bad surface chemistry or poor hybridization technique can sometimes be attributed to poor sample preparation. Microarray sample preparation requires a high level of sample purity to work well. Contaminates in the spotted material can inhibit the surface chemistry coupling reaction and can also clog micro spotting pins. The opening of a 946 micro spotting pin is ~20 microns, so the material being spotting must be smaller. All types of DNA, proteins, peptides and cell lysates are routinely spotted into microarrays using a variety of solvent and aqueous based buffer systems. When solvents used to precipitate or synthesize DNA get downstream into a sample, they ruin the surface tension properties of a sample making it difficult or impossible to spot consistently. If one sample is contaminated with a solvent, and the sample next to it is not, this virtually guarantees that those spots will be different sizes. This can be very problematic since microarrayers are programmed to spot at specific center-to-center distances and spots that are merged together ruin data analysis.

Once samples are prepared, they need to be added to a suitable printing buffer at the right concentration. An optimized printing buffer accomplishes many different tasks.

  • Print even, small, round spots
  • Disperse the sample evenly within the spot
  • Promote sample binding to the array surface
  • Retard evaporation within the source plates
  • Dry evenly once spotted
  • Wash away easily in processing
  • Optimize attachment to the microarray surface
  • Stabilize sample for prolonged storage
Depending on the sample and application, all of these requirements may not be necessary, and other characteristics may be desirable. For example, some DNA microarray scientists prefer to use a denaturing agent in their printing buffer to avoid the boiling, denaturing step prior to hybridization. However a denaturing agent in the printing buffer would be the demise of an antibody-based microarray. Additionally, some chemistries do not require the sample to be dry for coupling to the surface reactive groups. See some protocols for spotting buffer and sample preparation by clicking here.

5. Environment

The key elements of environmental control are cleanliness, temperature and humidity. There are two environments to control, one inside the chamber of the microarrayer and one outside the chamber. Cleanliness in and around the microarrayer is important for obvious reasons. Cleanroom level environments are considered essential for commercial level manufacturing, but are not essential for research. Many microarrayers are equipped to filter the air to clean room level quality inside the arraying chamber. The NanoPrint uses a unique liquid filtration system that both cleans and humidifies the air during print runs. The system can also dehumidify the air if the ambient conditions are too humid to print high density microarrays. Temperature and humidity are important to keep samples from evaporating during a print run. Samples must stay hydrated in the source plate as well as in the printing mechanism while they are being spotting The loading volume of a micro spotting pin is typically only 0.25 ul and hundreds of spots from that single pickup is desired, so humidity levels of at least 50% are used for spotting. Additionally, some microarray surfaces require long term storage in high humidity conditions to establish coupling of the sample to the substrate.

Get these 5 parameters dialed in and you'll never miss the spot. Contact me at for more information about microarray manufacturing. I also do consulting work if you want to have me visit your organization and troubleshoot existing manufacturing setups.