The geneCube is the ideal platform suitable for assaying a small to medium number of genes or proteins (e.g. 50 - 1,000) on a large number of samples (e.g. 100 to 1,000,000). Assaying a large number of samples is essential for generating the very large amount of information to realize the full potential in disease diagnosis, drug development, toxicological applications and environmental applications

Below follows a brief overview of our platform. For more detailed information download the following whitepapers: Plexigen Platform.pdf (1.1 MB) Plexigen Gene Expression.pdf (0.5 MB) Plexigen Protein.pdf (0.5 MB)

Plexigen's primary technology starts with the geneCard™, the fundamental unit of our microarray platform. Unlike other microarrays currently on the market, Plexigen's geneCards are truly three-dimensional(3-D). Plexigen's geneCards are injection molded commercial high-temperature vulcanized silicone rubber sheets about 4 cm by 6 cm, 1 mm thick and penetrated by 404 conical holes.

Picture of a 404 feature geneCard™.

The diameters of the large and small openings of the conical holes are 1 mm and 0.4 mm, respectively. The interior of these holes, also called features, is where the probes (DNA segments or proteins) are attached. The conical surface provides an area for immobilization of each probe of about 2.5 mm². This area is considerably larger than those commonly found in two-dimensional arrays, in which typical features range from 20 µm to 150 µm in diameter and have areas of 0.0003 - 0.018 mm².

Cut away view of a geneCard™

The geneCards are then stacked together, up to hundreds of cards high, to form a geneCube™. The assembled geneCube now has a number of assays equal to the number of geneCards stacked together.

Stacking of geneCards to form a geneCube

Using our Plexstation samples are then drawn through the 404 columns formed by the stack of geneCards.

Flow through a geneCube

After removing the samples the cards are then read with our scanner the geneReader

The power of a geneCube is that hundreds of samples, either genetic or proteomic, can be assayed simultaneously. For example a geneCube with 100 cards can genotype 100 genes for 384 samples with 20 controls in parallel. This unique feature puts Plexigen's product into a tremendously advantageous position over all currently available microarrays.

Listed below are some major advantages of Plexigen's family of microarray products and links to more information:

High throughput

Plexigen's microarrays have extremely high throughput. The throughput can be either with a large number of genes or a large number of samples. The following three examples are used to illustrate this point.

Example one: 225 3D geneCards can be stacked together to form a single cube using our current instruments. The chip density thus immediately reaches to 90,900 with 404-feature 3D geneCards and to 345,600 with 1536-feature 3D geneCards. This is more than enough to cover major sequenced genomes such as human, mouse, fruit fly, yeast, etc.

Example two: In pharmacogenomics or disease diagnostic studies, a set of surrogate markers have been identified. The goal is to use them to screen a large number of samples. With Plexigen's current cube, 404 samples can be assayed in one reaction simultaneously. 2000 patient's samples would only need 6 cubes.

Example three: Generic high-sample count example measuring the expression level of 200 genes in 10,000 samples. Assume one set of instruments is employed for the project.

See our direct services for a more detailed description of our pricing.

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Quality Control

Surface-based microarrays are coated with probes individually (one at a time). Besides a low production yield, another major disadvantage is the difficulty implementing rigorous quality control. A microarray will be destroyed when it is tested for its quality. Thus the quality of a single microarray is not representative of the quality of a batch of microarrays.

The geneCards are fabricated and coated in a quantity of a thousand at the same time under identical conditions. During the geneCard probe coating process, probe solutions are saturated on the wall of each feature in a geneCube simultaneously, thus allowing probes to chemically bond to the surfaces. The probes can be uniformly coated onto all the geneCards and features. A number of cards from each batch, or wells from each card can be used for purpose of rigorous quality control and the QC tested cards will be highly predictive of the quality of the entire batch.

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Integrated Information Management

Throughout the process of product development, Plexigen has always carefully and thoughtfully planned how to ensure data accuracy and smooth information flow. By constantly communicating with Plexigen's internal database, probe, target, individual geneCard and final cube identity are accurately determined. For example, each geneCard is identified with a 20 feature on-chip ID and each cube is identified by a smart memory chip.

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Dynamic Range

The seperate features with large surface areas are scanned individually with a PMT and illuminated with a laser. This arraingement eliminates cross-talk and results in true 24-bit data and effective dynamic ranges of:

> 4 log 10 for RNA targets and for protein targets
> 3 log 10 for direct and secondary antibody detection
> 5 log 10 for capture assay.

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Plexigen’s geneCard reaches much higher accuracy than the competing microarrays on the market by its uniform assay environment, much larger hybridization area and control of the flow of hybridization solution. The probes are immobilized on the inside surface of a cone shaped feature for Plexigen’s geneCard. The effective hybridization area for each probe in the Plexigen geneCard is ~2.5 mm² which is considerably larger than that for surface-based microarrays. For conventional microarrays, data for different samples are usually generated at different times and under different experimental conditions. A geneCube can generate experimental data for multiple samples in parallel, at the same experimental time and under identical experimental conditions. The experimental errors between samples are significantly reduced by using the geneCube. In an experiment involving measuring gene expression levels for 34 human organ samples, the coefficient of correlation between replications for all the samples over all the genes are above 0.97. The coefficient of variation among geneCards ranges from 0% to 10% with a median of 0.4% for log transformed signal intensity.

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>The geneCube is sensitive enough to accurately detect a target at 0.06 pM concentration in a control experiment with known target concentration. In this experiment, different amounts of labeled cRNA were used to hybridize to the features for some human samples. In a 40 µl hybridization solution, 1 µg, 0.5 µg, or 0.25 µg of total labeled cRNA was used. Significant differences between the three concentrations were observed for many combinations of genes and samples. This is direct evidence that the geneCube platform is sensitive enough to resolve gene expression differences of less than two fold.

Using a generalized linear model to analyze the data, results have shown that the geneCube platform can resolve a 1.4 fold gene expression difference with four replications. Because of its flexibility in experimental design and straightforward way of producing many replications, the geneCube platform can resolve very small differences in gene expression.

The geneCube platform has a sensitivity of detecting 0.06 pM of target in solution, which is sufficient for most applications in genetics-based clinical diagnostics. 99.6% of the signal intensity variation attribute to cRNA concentration.

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Reproducibility

Accurate readings from hybridization assays are essential for a meaningful comparison between biological samples. The stacked substrate cube can potentially reach much higher accuracy than 2D surface-based hybridization substrates because of its uniform assay environment, a much larger hybridization area, and the ability to control of flow of hybridization solution. Data for different biological samples are usually generated on two-dimensional substrates at different times or under different experimental conditions, because of the limited number of samples that can be analyzed on a single substrate. A geneCube of stacked substrates can generate experimental data for multiple samples in parallel and under the same experimental conditions.

Coefficient of correlation between replications for samples based on values across 40 genes where each gene was replicated between 4 and 12 times combining replications across features and geneCards. The coefficient of correlation is higher than 0.97 for all cases.

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Flexible Experimental Design

A good experimental design is an important first step for any experiment. Many designs require replications at the gene level, chip level or even sample level. This is simply not feasible in terms of cost and efficiency with current state-of-the-art microarrays. On the contrary, Plexigen's geneCubes are well suited for any flexible experimental designs which ensures the validity for any formal statistical significance testing. Pharmaceutical industry and research institutions have not had this capability in micrarrays until now with the Plexigen system.

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Small Amount of Sample

The geneCube platform only needs a small amount of target solution for assaying all the geneCards. Each feature holds 0.24 µl solution. For example, 40 µl of reagent will fill one feature for a geneCube with 166 geneCards. We have used a total of 0.25µg label RNA diluted into 70 µl of hybridization buffer to hybridize into each channel and got repeatable results. The platform might not need an amplification step for gene expression-based applications. This advantage will also enable many high throughput screenings and practical clinical applications using proteomics.

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Suitable for Many Applications

The geneCube is a platform technology which is suitable for many types of assays. We have generated a significant amount of data for applications in gene expression, protein interaction and genotyping.

Several versions of traditional ELISA assays for detection of protein interactions, such as antibody-antigen interactions, have been implemented on the geneCube platform. The platform has very high sensitivity and reproducibility for the assays. The platform can be used for high throughput proteomics experiments, such as protein-protein interactions, protein-small peptide interaction, antibodies screening for antigens, and antigens screening for antibodies.

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Reduces Liquid Handling

Being compatible with established liquid handling instruments is a great advantage of Plexigen's geneCubes. The geneCube can be loaded with samples directly from standard microtiter plates using only one axis of motion. No additional liquid handling is needed once the samples are processed.