Presynthesized microarrays can be created by the microdeposition of various biological compounds onto a substrate. The presynthesized biological materials can include oligonucleotides, cDNA, enzymes, antibodies, various proteins, human serum, microspheres and even cells. The substrate material can be comprised of glass slides, nitrocellulose or PVDF membranes, acrylamide, polystyrene, cellulose, or other acceptable substrate material.
The number of different and important microarray applications is impressive. Gene expression profiling is the most notorious microarray application in which the expression of multiple genes controlling protein synthesis in normal and abnormal tissues is compared and interpreted. Another microarray application is toxicology analysis for viewing the effect a compound has on increasing the expression of genes associated with toxicity in organs, such as the liver. De Novo gene sequencing is a high-throughput application to perform sequencing by hybridization using a microarray containing a large number of oligonucleotides. Antibody microarrays are important tools for the investigation of the progression and regulation of tumor cell development. Cancer antibody microarrays containing a diverse panel of monoclonal antibodies, such as for Mitosin, ATM, BRCA1, Retinoblastoma (RB) Protein, Topoisomerase II, CA-125, etc., can be used for research of tumor cell proliferation, suppression, and drug resistance.
“Capture” microarrays typically utilize antibodies for the ligand-binding. These microarrays may also use peptides, nucleic acid aptamers or other alternative protein scaffolds for the detection of molecules in mixtures, such as plasma or tissue extracts. For diagnostics the capture microarrays can be used to carry out multiple immunoassays in parallel by testing for several analytes in individual sera or testing many serum samples simultaneously. In proteomics, capture microarrays are used for protein expression profiling to compare and quantitate the protein levels in different samples that represent healthy and disease states. These protein expression microarrays may become a complement to the current 2DE protein separation technology. Protein microarrays may also be used to correlate the polymorphic changes resulting from SNPs via protein function. In vitro functional interrogation microarray screens can be used to explore analyte-ligand interactions, such as protein-protein, protein-DNA, protein-drug, receptor-ligand, enzyme-substrate, etc.
The method of spotting microarrays is achieved traditionally using a computer-controlled xyz motion stage with a head carrying a pen device to pick up small drops of solution from the multiwell plates for transfer and spotting them onto a surface. These spotting pens are sophisticated designs adapted from the quill type of ink pen. The pen printing is reliable and repeatable when using a flat solid surface substrate. Problems can arise with the contact technology when using uneven and membrane types of substrates. The uneven substrates can result in missed spots when the surface regions are lower than the level of a pen or pens within a bank of printing pens. Spotting onto membranes can result in unacceptable surface indentations and uneven spotting if the membrane absorbs the spotting solution too quickly. Other disadvantages include the limited range of volume control for each spot printed and the inability to overprint without the risk of cross contamination of the spotted fluids.
Positive pressure displacement is another spotting method that utilizes a syringe system or valve jet for deposition of the fluids. In valve-jet technology an orifice or nozzle is attached to a solenoid valve that opens and shuts rapidly to produce streams of intermittent droplets from a pressurized flow. A syringe system picks up the fluid from sample wells and then dispenses the fluid onto the substrate using positive displacement. These systems are highly reliable, as the fluidic property influence on dispensing is less than the effect on piezoelectric microdispensing. However, these positive pressure displacement microdispensing systems can have lower repeatability rates when dispensing at their lower volume capabilities. The low-end deposition volumes for these systems are in the nanoliter range.
MicroFab Technologies, Inc. produces drop-on-demand piezoelectric microdispensing devices. In this type of microdispensing device, the fluid is maintained at ambient pressure and a piezoelectric transducer is used to create a drop only when needed. The transducer creates a volumetric change in the fluid resulting in pressure waves. The pressure waves travel to the orifice, are converted to fluid velocity, that results in a drop being ejected from the orifice.
As a non-contact printing process, the accuracy of ink-jet dispensing is not affected by how the fluid wets a substrate, as is the case of positive displacement or pen transfer systems “touching off” the fluid onto the substrate during the dispensing event. Thus, the fluid source cannot be contaminated by fluid already on the substrate or by the substrate material. Therefore, it is possible to overprint spots using a different reagent or biofluid without the risk of cross contamination. Finally, the ability to free-fly the droplets of fluid over a millimeter of more allows fluids to be dispensed into wells or other substrate features (e.g., features that are created to control wetting and spreading).
MicroFab Technologies is using single glass tube piezoelectric dispensers (see Equipment section), but has also focused on adapting its high density drop-on-demand array printhead technology to bioactive fluid microdispensing. The use of an integrated array ink-jet printhead simplifies both the design and operation of multiple ink-jet systems, particularly when high density/accuracy is required.
Left: ten fluid integrated piezoelectric drop-on-demand array printhead prototype. Right: results from ten fluid printhead operation: eight different dyes printed onto 200um centers.
The use of piezoelectric drop-on-demand ink-jet printing technology for microdispensing fluids has broad applicability in DNA and immunoassay diagnostics, expression studies, and high-throughput screening. Using ink-jet microdispensing technology, spheres of fluid with diameters of 25-100um (10pl to 0.5nl) can be produced at rates of 0-4,000 per second. Multiple microdispensing devices can be used to print multiple fluids (probes, reagents, bio-sample fluids, surface activation fluids, etc.).
Left: 250um diameter spots of oligonucleotide probes complimentary to drug resistant Mycobacterium tuberculosis DNA sequences printed onto a glass microscope slide [Collaboration with Houston Advanced Research Center]. Right: 100um diameter spots of seven k-ras DNA probes and a biotinylated marker, printed on 500mm centers, and hybridized to a mixture of seven complimentary k-ras target sequences [Image courtesy of Beckman Instruments].
Left: 100um diameter spots of oligonucleotide probes complimentary to wild type Mycobacterium tuberculosis printed in an array onto nitrocellulose to create a negative outline of the “TB” pattern. Right: 100um diameter spots of oligonucleotide probes for Mycobacterium tuberculosis Rifampicin resistance rbo-ß gene mutation printed in an array onto nitrocellulose to create a positive “TB” pattern.
The simultaneous detection of a plurality of nucleic acid targets using arrays of immobilized sensor probes is a useful tool for molecular diagnostics. Resistance to Rifampicin, a first line drug for tuberculosis, which is carried on the rpo-ß-gene of mycobacterium, has been chosen as lead parameter. The potential to resolve point mutations at the presence of pronounced formation of strong intra-strand secondary structures and extremely GC-rich segments has been shown.
Mycobacterium tuberculosis Rifampicin resistance chip containing 11 different 18-mer oligonucleotide sensor probes, plus control oligonucleotide spot for monitoring the conjugate and detector performance [Image courtesy of Roche Diagnostics (formally Boehringer Mannheim)].
Microspot antibody and peptide arrays can be fabricated using ink-jet technology in a similar manner as DNA arrays. These can be used for clinical diagnostics or proteomic analysis (protein expression studies).
Sandwich immunoassay microarray with detection of different nanomolar concentrations of Cytochrome C using rabbit anti-cytochrome C; spots are 40um in diameter.
Microspot arrays can also be comprised of larger volume spots by the accretion of microdrops onto the substrate. A 4x5 array of 250mm diameter spots of resorufin labeled streptavidin printed on a 500mm pitch onto a glass microscope slide. A 2.5nL volume (25 drops) was deposited for each spot [Image courtesy of Beckman Coulter].
Ink-jet printing of proteins was demonstrated in the early 1980’s. In one application, patterns of antibodies were printed onto membranes, typically nitrocellulose, that bound the antibody for use in diagnostic assays. The image shows Abbott’s Pregnancy indicator TestPack™ that has nitrocellulose printed with two antibodies (typically, ßHCg and a control) using a two fluid continuous ink-jet printing system developed by MicroFab Technologies. The Abbott TestPack™ was also available for streptococcus and drugs of abuse testing.