Thursday, October 25, 2007

Microarray Slides

Many studies have been done to test what microarray surface is best for DNA and protein microarray applications. The principles that determine how microarray surface chemistries are used and interpreted are as follows:

Rule 1. Attach biomolecules in a stable manner.

A stable attachment is key to producing strong signals and accurate results. Capture molecules must be immobilized, remain in a specific location and essentially stay unchanged throughout all the pre and post printing procedures of the microarray experimental life cycle. Only a stable surface can meet this need.

Rule 2. Maintain activity of attached biomolecules

The binding, hybridization, epitope and enzymatic activity properties of biomolecules are essential to usability of the microarrays. Without the binding activity of the printed sample, there can be no experiment. Activity can be prolonged on a surface by using stabilizing printing buffers; therefore the activity of a biomolecule on a surface is not completely dependent on the surface chemistry.

Rule 3. The surface binding mechanism must have longevity.

The surface used in a microarray experiment must maintain its binding activity for at least several months, in some cases years. Time and flexibility is required to design experiments, plan manufacturing, procure product, prepare samples and store printed microarrays prior to binding reaction, data acquisition and analysis. Shelf life for a microarray after biomolecules have been spotted and attached can be years under proper storage conditions. That same surface may need to be spotted onto within several months after the surface chemistry has been made for best results.

Rule 4. Accurate biological representation of each sample

The density of reactive groups and/or attachment method on the surface must be uniform across the entire substrate to ensure identical coupling of biomolecules at each microarray location. The molecules attached to the surface must reflect an accurate representation of the sample from the source plates. Samples must not be allowed to attach to the source plate and reach the surface chemistry at the proper concentration. Lateral flow of the sample on the surface must be kept at a minimum to keep spots defined and the density of bound molecules within a spot to optimize reaction kinetics. "Too much of a good thing" is true when it comes to microarray spots...too little will compromise results as well. The surface must be completely homogenous. Without homogeneity, different amounts of biomolecule will attach in different areas of the substrate and compromise quantitation. Regardless of the printing technology (contact or non-contact), the samples being printed at a spot location are being deposited in saturating volumes to the attachment sites of each location. What does not attach to the surface in each spot washes away in the pre-processing steps of a microarray.

Rule 5. A microarray surface must be planar.

Surface flatness is important to achieve high quality printing and detection. Accurate robotics and sophisticated printing devices are used to deposit array elements. That means the distance between the printing surfaces and printing mechanism must be calibrated within a minimum of tens of microns for high quality printing to take place. Surface roughness, parallelism, and flatness are all important characteristics. Unlike contacting printing with the 946 and Stealth Micro Spotting Devices, spot size of non-contact delivery systems is determined by how far away the delivery nozzle is from the printing surface. In other words, the farther a sample travels through the air, the bigger the spot gets. Surfaces that deviate in height will compromise spot quality. Additionally current microarray scanners have focal planes in the resolution range 5 to 50 microns. Accurate planarity of the substrate is required to maintain proper focus for acquisition. This is not a problem if your detection instrument as dynamic auto focus...many microarray detection instruments do not have this feature. Reacted microarrays must stay in focus during scanning and the physical properties of said substrate and reacted immobilized array elements should not change during or after scanning.

Rule 6.The surface must have low intrinsic background noise.

Fluorescent detection instruments dominates the microarray industry, however, colorimetric, chemiluminescent, light scattering, surface plasmon resonance, planar wave guides and others are being implemented. Regardless of the detector, the surface must not significantly contribute to the background noise of the detection system. For example, surfaces such as non-reflective clean white membranes read as zero background on the SpotWare Colorimetric Scanner. Glass is the preferred material for fluorescent-based detection devices, since white surfaces reflect photons into the PMT or CCD detector and cause high background.

Rule 7. Standard physical dimensions of the substrate are required.

The same physical size of each substrate facilitates automation for manufacturing and processing....thus dramatically increasing microarray usability. The standard in the microarray industry is 25 mm x 76 mm x ~1 mm. Affymetrix is one of the few examples of successful implementations of microarray technology outside this standard format. ArrayIt, Agilent, NimbleGen and many other organizations has standardized on the microscope size slide glass format. A standard format assures compatibly with open microarray platforms, reduces cost, promotes innovation and increases flexibility for the end user by empowering the use of products from multiple vendors.

Rule 8. Substrates require lot-to-lot, piece-to-piece consistency.

Each substrate must be the same to guarantee accurate results. A surface used in an experiment must work the same from one day to the next, one experiment to the next. Without consistency, it is impossible to plan, design, execute, analyze and compare experiments over time.

Rule 9. A microarray surface must be amenable to mass production

A lab running a microarray test could use dozens to hundreds of microarrays per day. That means that only one lab and one application requires ~25K substrates per year. Any surface must meet all the rules presented here and allow for mass production rates to guarantee availability. Surfaces prior to and after sample immobilization that do not require special storage conditions and have a long shelf life will be most desired. The ability for mass production allows for economies of scale, which leads to the most important characteristic, the product be affordable.

Case Study: Adhering to the rules

Our surface chemistry product development efforts adhere to this list of criteria. Our SuperEpoxy 2 surface is a good example. This surface is designed to bind biomolecules via free primary amine, thiol and hydroxyl groups covalently...making it useful for DNA, Protein and Peptide microarray applications. This type of reactive surface has tested favorably when compared to other 2D and 3D coupling strategies (see comparison below). To make the product we start with sheets of glass and manufacture it to be 25mm x 76mm x 0.96 mm size. Thickness is 0.96 mm for the reason that some of the surface of the glass is removed during our high precision polishing process. This polishing process makes the surface of the glass atomically homogenous. This homogeneity has been confirmed by AFM.

The surface is then cleaned, sterilized, and activated in class 100 cleanrooms. The surface is made active with primary amine binding reactive epoxide groups with covalent silane chemistry. Our precision in, precision out approach that takes advantage of the atomically homogenous glass and covalent chemistry assures our end result is homogenous. Without a perfect homogenous glass surface to start with, any chance of achieving a homogenous surface after silanization is lost. Our covalent approach allows us to easily remove reactive groups that do not bind in the activation process, leaving a pristine homogenous surface. It is an important to note the distinction between a treated and coated surface. A coating is a monolayer on top of the glass, and a treatment completely modifies the glass. All manufacturing processes are performed in a cleanroom setting to assure strict environmental control to further improve cleanliness and lot of lot consistency.

Advantages of 2 dimensional surface chemistry for microarray
(activated homogeneous glass)

  • –Better defined spot morphology (no diffusion)
  • –Inherent lower background (glass)
  • –Compatible with SPR, planar wave guides, RLS and other exotic detection strategies
  • –High specificity of binding
  • –Non-porous surface (no place to trap any contaminate in processing) –Covalent and/or specific binding to avoid altering biological activity

Advantages of 3 dimensional surface chemistry for microarray

(membranes, filters & gels)
  • –High binding capacity (absorption)
  • –Compatible with fluorescent, chemiluminescent, and colorimetric detection
  • –Longer history of use (comfort level for users such as nitrocellulose, nylon and PVDF
  • –Less expensive labeling reagents and reading equipment (colorimetric) –3D capture to avoid altering biological activity

If you would like to discuss microarray surface selection, please contact me. 408-744-1331, .