Microarray Immunoassay Experimental Design

Arrayit's microarray platform is highly flexible and powerful suite of technologies for miniaturizing and multiplexing existing assays such as those for gene expression, genotyping, immunoassays and others.
Microarray immunoassay test development requires a little work up front, but it's fun to do. Microarrays are highly affordable and can reduce the cost of running immunoassays, we welcome the opportunity to manufacture custom microarrays to meet almost any experimental need. Here is a list of questions to help guide the process of experimental design. You don't have to answer all these questions to have a legitimate project, questions are not applicable to all projects. We sign confidentiality agreements, we don't expect customers to disclose things they do not wish to disclose! Contact me at todd@arrayit.com, 408-744-1331 with any questions.

1. How will the microarray assay be used and what are the goals of the experiments or tests?
2. What is the specific biomolecule or analyte that you want to measure?
3. Have ELISA assays been run with the sample types?
4. What capture analytes are we printing into a microarray?
5. What are the characteristics of the capture analytes for the microarray?
6. Antigen, antibody, cell lysate, peptide...other?
7. If antibodies, do you have a secondary antibody for each capture antibody?
8. Amount of purified capture analyte available?
9. If the analytes are peptides do you have all the amino acid sequences?
10. Are there any safety issues associated with the handling or use of the analytes?
11. Storage buffer (including pH)?
12. Soluble in water or is another buffer required?
13. Storage conditions?
14. If analyte is lyophilized, were any stabilizers used?
15. What is the origin of the sample to be tested...blood, serum, plasma, water, tissue culture supernatants...?
16. Do the samples to be tested on the microarray have to be pre-treated, such as extractions or centrifugations? If yes, what are they?
17. What is the expected outcome of the immunoassay you want to develop?
18. Are we developing a microarray assay that will require routine testing?
19. How many samples will be tested overall?
20. How many samples do you want to test for at one time?
21. Do you require automation, or is manual processing OK?
22. Did you want to outsource the testing or do parts or all of it?
23. Do you want to be empowered to make your own microarrays, or have us do it for you? (costs less if we do it).
24. Do you have a microarray scanner or other instrument for reading the test?
25. Are you planning fluorescent, colorimetric, chemiluminescent, SPR or other detection platform?
26. Does your test require FDA approval (or will it in the future)?
27. Is there anything else important you think we should know?

http://www.arrayit.com/Services/Protein_Microarrays/protein_microarrays.html

Microarrays

Arrayit's microarray technology empowers the arrangement of a miniaturized array of test sites or "spots" on a planar surface. Each spot is typically around 100 micrometers in diameter , but they can be larger and smaller as well. With our platform the microarrays can contain a wide variety of biological molecules, but most often they are some form of DNA or protein and the planar surface is most often a specially prepared glass slide. The biomolecules on the slide act as targets for a test sample applied in solution to the microarray. Arrayit's microarray format allows many tests or experiments to be performed simultaneously, miniaturized and in parallel, leading to the generation of huge amounts of biological information for the application of only a tiny amount of test sample.Microarrays can be put into a larger catagory called "biochips" with microarrays being the main technology component of a microfluidic testing device.The purpose of this blog entry is to review how Arrayit's technology fits into the overall market aimed at analysis of proteins and DNA.

Current applications include:
1) expression profiling;
2) genotyping
3) cytogenetics
4) serum-based diagnostics;
5) protein-protein binding assays;
6) dna-dna binding assays;
7) drug-target binding
8) receptor epitope binding
9) small molecule HTS

Arrayit has a uniqueset of products in both DNA and protein array testing equipment and supplies using screening reagents and instruments for analysis of individual components of cells, body fluids, blood, serum or plasma. Arrayit has sales worldwide with tremendous growth potential in the area of diagnostics. Arrayit products are used in research labs, hospital labs, and commercial laboratories.

The main technology driver of the Arrayit platform is our ex-situ microarray manufacturing technology. This is the cornerstone of our platform that is able to make more microarrays with less sample, faster than any other technology in the world.

- How can Arrayit's microarray tools and technologies facilitate drug discovery, design and development?

Answer: By providing specific functional information about genes and proteins drug designers can have reliable and measurable targets for their therapeutics with the ability to measure efficacy.

- What are the main types of microarray technologies that are currently available?

Answer: The most reliable and versatile type of microarray is made with Arrayit's ex-situ technology. The reason for this is only through an ex-situ technique can the biomolecules being used to manufacture the microarray be checked for quality prior to the microarray manufacturing step. In-situ manufactured microarrays can only be checked for quality after the in-situ manufacturing step and in-situ microarrays are limited to synthetic biomolecules only.

- Who are the current key players in this marketplace?

Answer: Arrayit has been a key player in the research market for more than a decade. Arrayit's platform is now slated to be used in clinical diagnostic applications.

- Which microarray market areas have the greatest potential for growth?

Answer: Diagnostics

- What is the current state of the microarray market?

Answer: Use of protein and peptide microarrays is increasing. Many end users have become more sophisticated, with a significant trend in outsourcing. Most doctors and scientists simply want accurate data, fast and at a good price. ...they don't care how the correct answer is generated.

- Which biotechnology and pharmaceutical companies are investing in microarraysolutions?

Answer: All major research institutions, biotech and pharmaceutical companies use microarray technology.

- What are the main microarray business strategies adopted by leading companies?

Answer: Depends on what assay is being targeted. Unlike other microarray companies, Arrayit's platform is extremely flexible in the types of applications that can be done. This leads to a broad marketing strategy and opens up more opportunties in diagnositcs.

- What are the benefits of Arrayit's microarray technology platform?

Answer: The ability to quantitively measure any type of biomarker including, mRNA, DNA, Protein, Peptide, and Carboydrate in a microarray format.

Personalized Medicine

On Jan. 19th and 20th we presented our microarray technology to ~400 attendees interested in the future of personalized medicine applications. After attending this event, it is clear to us that Arrayit technology is ahead of the competition in several key areas. The areas include high throughput genotyping, protein/peptide microarrays, and measuring gene expression.

The most interesting talk was presented by Dr. Elizabeth Mansfield. She is the Director of the Personalized Medicine Staff in the Office of In Vitro Diagnostic Devices in the Center for Devices, FDA, where she is developing a program to address companion and novel diagnostic devices. Hearing details about the regulations that are being implemented to protect the public health gives us a lot of confidence that our technology is best suited to meet the needs of our healthcare system.

It was a great event and we look forward to our work in this area. If you have any questions about how our technology is being implemented in personalized medicine applications, please contact me at todd@arrayit.com

Parkinson's Disease Diagnostic Development

Not long ago I donated a fibroblast sample from my upper arm so that my cells could be cultured and my DNA could be a "normal" control in a microarray experiment. At least someone thinks I am normal!! This was done in conjuction with our research collaboration with the Parkinson's Institute to developed a molecular diagnostic test for Parkinson's Disease. President Barack Obama said, "It took a lot of blood, sweat and tears to get to where we are today, but we have just begun. Today we begin in earnest the work of making sure that the world we leave our children is just a little bit better than the one we inhabit today." I'd like to add it takes a skin fibroblast sample too! This is for development purposes only, final goal of the test we development will not require a patient to provide a fibroblast as shown here.

Area was made numb by a topical anesthetic, then sterilzed with both an alcohol and iodine. Didn't hurt a bit. A special tool was used to make sure a perfect size little circle was cut.

The fibroblast sample is at the end of the pair of tweezers.

The hole in my arm was patched up using a special tape that stays on like skin. I am happy to say that it hardly left a mark on my arm and things are healing up very nicely.

Fibroblast sample went into a special buffer and media to make sure that the cells say alive and go into a special culturing procedure immediately. Sample preparation is a very important step. Our microarray platform works so efficiently that it is possible to detect changes in data based on how the samples are prepared. In any scientific comparative study, each and every sample should be handled as indentically as possible.

If you want to learn more about our collaboration with the Parkinson's Institute, please contact me http://www.arrayit.com/ or todd@arrayit.com .

Microarray Hybridization Automation

This is a short review of the mixing techniques used in microarray binding reactions.

Current micro-fluidic mixing technologies that are being implemented in the microarray industry include, Turbulent Flow, Rotary Mixing with air bubbles, Laminar flow, Acoustic waves, Surface Acoustic Waves, and Chaotic Mixing. The goal of mixing a microarray binding reaction is to assure that every molecule in solution finds its binding partner immobilized on the microarray as quickly as possible. In other words, the most desirable binding reaction has fast and complete diffusion of all biomolecules in solution over all microarray spots and remains a homogeneous mixture until the reaction is complete.

Sound microarray data can be achieved using inexpensive hybridization cassettes, however, in certain cases active mixing has been shown to speed up binding reactions, improve data quality, and reduce the number of molecules required in solution for the binding or hybridization reaction. Reasons to use automated machines for microarray binding reactions include:

• Save time and money by performing tests faster with less test sample
• Minimize handling of microarrays, which reduces the possibility of human error.
• Get better control over the experimental variables, resulting in increased reproducibility
• Empower users to define, edit and store individual methods and protocols
• Save and link experimental or testing procedures to database

In a turbulent flow system, the hybridization cocktail is mixed by the using random contact with the physical structure of a reaction chamber. One challenge of this type of system is obtaining homogeneity of the reaction mixture.

Rotary mixing with air bubbles is performed inside a sealed reaction cassette by rotating a trapped air bubble over the microarray. Binding reactions cannot take place in air, only in solution. Therefore a challenge of this approach is minimizing air bubble-based oxidization of the fluorescent dyes commonly used in microarray reactions, which would lead to lower signals and elevated background. Another challenge is that if an air bubble were to become trapped, the reaction in the trapped area would not proceed.

Laminar Flow is generated by using a small diaphragm pumping system at each end of the microarray to move the binding reaction sample back and forth across the microarray. Recent micro-fluidic studies show that laminar flow mixing can produce layers of liquid that flow over top of each other, thus one challenge of this approach is to obtain fully homogeneous and complete mixing of sample during the laminar flow process.

Surface Acoustic Waves generated by piezoelectric transducers are used to cause streaming of the hybridization reactions under cover slips or lifter slips. Some of the same challenges that apply to laminar flow also apply to systems that use surface acoustic waves.

Chaotic Advection Mixing is accomplished using micro fluidic pumps and a mixing loop. The overall movement of liquid in properly configured systems is “chaotic” due to the extremely complex direction and speed of fluids provided by the mixing loop. This type of system provides the most complete mixing of low volumes of liquid in the shortest amount of time. For more information on this patented microarray processing technique please view the product page by clicking here.
Below is a movie showing how our system takes a fluorescently labeled biomolecule in solution applied to a microarray, and mixes it completely over the entire microarray in less than 30 seconds. The liquid remains mixed throughout the reaction. The bright white dot is the injection port area for the hybridization cocktail...watch how nicely the reaction reaches equilibrium.



If you are evaluating systems, ask your vendor to provide a movie showing physical evidence that they can provide good mixing! I give special thanks to Dr. Djamel Bouraya for providing this video and other information for this blog entry.


For more information, contact me at todd@arrayit.com or at http://arrayit.com/

Biomarker Panel Microarrays

Reducing the cost of assays is always a good thing. With this great new tool we miniturize, multiplex and parallelize existing assays. We eliminate evaporation and perform 24 separate microarray reactionss on a single slide substrate. Our ex-situ microarray manufacturing method allows us to put hundreds of genes, proteins or other biomolecules in each reaction well. This allows us to amortize our surface chemistry cost over 24 separate microarray experiments, reducing cost by a factor of 24. It also increases throughput and is very easy to use. see http://www.arrayit.com for more information.

Ovarian Cancer Diagnostics

As reported today in numerous sources, Arrayit Diagnostics licensed ovarian cancer biomarkers from Wayne State University. Our test will aim to effectively screen for early-stage ovarian cancer in women who are not yet symptomatic, addressing the fact that the disease often presents with largely non-specific symptoms during its initial stages, and is diagnosed only when later-stage symptoms manifest and the disease has metastasized to other parts of the body. For more information see www.arrayit.com