EE-Unit-V Biochips

A biochip is a collection of miniaturized test sites (microarrays) arranged on a solid substrate that permits many tests to be performed at the same time in order to achieve higher throughput and speed. Typically, a biochip’s surface area is no larger than a fingernail. Like a computer chip that can perform millions of mathematical operations in one second, a biochip can perform thousands of biological reactions, such as decoding genes, in a few seconds.

A genetic biochip is designed to “freeze” into place the structures of many short strands of DNA (deoxyribonucleic acid), the basic chemical instruction that determines the characteristics of an organism. Effectively, it is used as a kind of “test tube” for real chemical samples. A specially designed microscope can determine where the sample hybridized with DNA strands in the biochip. Biochips helped to dramatically accelerate the identification of the estimated 80,000 genes in human DNA, an ongoing world-wide research collaboration known as the Human Genome Project. The microchip is described as a sort of “word search” function that can quickly sequence DNA.

In addition to genetic applications, the biochip is being used in toxicological, protein, and biochemical research. Biochips can also be used to rapidly detect chemical agents used in biological warfare so that defensive measures can be taken.

The notion of a cheap and reliable computer chip look-alike that performs thousands of biological reactions is very attractive to drug developers. Because these chips automate highly repetitive laboratory tasks by replacing cumbersome equipment with miniaturized, microfluidic assay chemistries, they are able to provide ultra-sensitive detection methodologies at significantly lower costs per assay than traditional methods—and in a significantly smaller amount of space.

At present, applications are primarily focused on the analysis of genetic material for defects or sequence variations. Corporate interest centers around the potential of biochips to be used either as point-of-care diagnostics or as high-throughput screening platforms for drug lead identification. The key challenge to making this industry as universally applicable as processor chips in the computer industry is the development of a standardized chip platform that can be used with a variety of “motherboard” systems to stimulate widespread application.

Historical perspective

It is important to realize that a biochip is not a single product, but rather a family of products that form a technology platform. Many developments over the past two decades have contributed to its evolution.

In a sense, the very concept of a biochip was made possible by the work of Fred Sanger and Walter Gilbert, who were awarded a Nobel Prize in 1980 for their pioneering DNA sequencing approach that is widely used today. DNA sequencing chemistry in combination with electric current, as well as micropore agarose gels, laid the foundation for considering miniaturizing molecular assays. Another Nobel-prize winning discovery, Kary Mullis’s polymerase chain reaction (PCR), first described in 1983, continued down this road by allowing researchers to amplify minute amounts of DNA to quantities where it could be detected by standard laboratory methods. A further refinement was provided by Leroy Hood’s 1986 method for fluorescence-based DNA sequencing, which facilitated the automation of reading DNA sequence.

Further developments, such as sequencing by hybridization, gene marker identification, and expressed sequence tags, provided the critical technological mass to prompt corporate efforts to develop miniaturized and automated versions of DNA sequencing and analysis to increase throughput and decrease costs. In the early and mid-1990s, companies such as Hyseq and Affymetrix were formed to develop DNA array technologies

The availability of genetic sequence information in both public and corporate databases has gradually shifted genome-based R&D away from pure sequencing for sequencing’s sake and toward gene function–oriented studies. It soon became apparent to everyone involved in genomics that gene sequence data alone was of relatively little clinical use unless it was directly linked to disease relevance. This, in turn, has driven the development of the field of pharmacogenomics—an approach that seeks to develop drugs tailored to individual genetic variation (see Pharmacogenomics, pp. 40–42).

In this regard, DNA-based biochips are at present used primarily for two types of analysis. First, they have been used successfully for the detection of mutations in specific genes as diagnostic “markers” of the onset of a particular disease. The patient donates test tissue that is processed on the array to detect disease-related mutations. The primary example of this approach is the Affymetrix GeneChip. The p53 GeneChip is designed to detect single nucleotide polymorphisms of the p53 tumor-suppressor gene; the HIV GeneChip is designed to detect mutations in the HIV-1 protease and also the virus’s reverse transcriptase genes; and the P450 GeneChip focuses on mutations of key liver enzymes that metabolize drugs. Affymetrix has additional GeneChips in development, including biochips for detecting the breast cancer gene, BRCA1, as well as identifying bacterial pathogens. Other examples of biochips used to detect gene mutations include the HyGnostics modules made by Hyseq.

A second application for DNA-based biochips is to detect the differences in gene expression levels in cells that are diseased versus those that are healthy. Understanding these differences in gene expression not only serves as a diagnostic tool, but also provides drug makers with unique targets that are present only in diseased cells. For example, during the process of cancer transformation oncogenes and proto-oncogenes are activated, which never occurs in healthy cells. Targeting these genes may lead to new therapeutic approaches. Examples of biochips designed for gene expression profile analysis include Affymetrix’s standardized GeneChips for a variety of human, murine, and yeast genes, as well as several custom designs for particular strategic collaborators; and Hyseq’s HyX Gene Discovery Modules for genes from tissues of the cardiovascular and central nervous systems, or from tissues exposed to infectious diseases.

Besides these two immediate array-based applications for this technology, a number of companies are focusing on creating the equivalent of a wet laboratory on a chip. One example is Caliper’s LabChip, which uses microfluidics technology to manipulate minute volumes of liquids on chips. Applications include chip-based PCR as well as high-throughput screening assays based on the binding of drug leads with known drug targets.

Finally, in addition to DNA and RNA-based chips, protein chips are being developed with increasing frequency. For example, a recent report describes the development of a quantitative immunoassay for prostate-specific membrane antigen (PSMA) based on a protein chip and surface-enhanced laser desorption/ionization mass spectrometry technology¹.

Industry challenges

A key challenge to the biochip industry is standardization. Both the assays and the ancillary instrumentation need to be interfaced so that the data can be easily integrated into existing equipment. This is particularly important when genetic diagnostic applications are at stake, because important clinical decisions are to be based on the interpretation of gene chip readouts, and these results need to be independent of the manufacturer of the biochip.

An example of an effort to address this issue is the formation of the Genetic Analysis Technology Consortium (GATC) by Affymetrix and Molecular Dynamics². The aim of this group is to establish an industry standard for the reading and analysis of many types of chips. In debating whether or not to join this consortium, companies are forced to decide whether their market niche will be broad use across the industry or highly customized applications in niche areas. When the decision is for the latter, it is unlikely that they will spend the time or money to standardize their product.

There are also important technical challenges for this industry that are fueling a highly competitive R&D race in order to establish market dominance. This is especially true in the “reader” technology to detect and decipher biochip readouts. Despite efforts to standardize this technology, novel platforms are being developed that promise higher throughput. One technology is that appears to have particular promise is the “optical mapping” of DNA. This method involves elongating and fixing DNA molecules onto derivatized glass slides in order to preserve their biochemical accessibility. It has the added feature of being able to maintain sequence order after enzymatic digestion. This system has shown promise for high throughput and accurate sequence analysis when integrated with appropriate detection and interpretation software³. Whether it will emerge as the system of choice, however, remains to be determined.

Finally, it is sometimes asked whether mass spectrometry can be part of next-wave biochip technology. As currently conceived biochips are essentially immobilized arrays of biomolecules, whereas mass spectrometry can determine molecular structure from ionized samples of material. Therefore, it is difficult to envisage a direct connection between the two, but perhaps in the future certain aspects of biochip analysis might be performed by mass spectrometry approaches.

Future directions

Biochip development will benefit increasingly from applications developed for other industries. For example, flame hydrolysis deposition (FHD) of glasses has many applications in the telecommunications industry, and is now also being applied toward the development of new biochips. A recent report describes how FHD was used to deposit silica with different refractive indices, resulting in microstructures that can be readily incorporated onto a chip and that integrate both optical and fluidic circuitry on the same device⁴.

Biochips are also continuing to evolve as a collection of assays that provide a technology platform. One interesting development in this regard is the recent effort to couple so-called representational difference analysis (RDA) with high-throughput DNA array analysis. The RDA technology allows the comparison of cDNA from two separate tissue samples simultaneously. One application is to compare tissue samples obtained from a metastatic form of cancer versus a non-metastatic one in successive rounds. A “subtracted cDNA library” is produced from this comparison which consists of the cDNA from one tissue minus that from the other. If, for example, one wants to see which genes are unique to the metastatic cancer cells, a high density DNA array can be built from this subtractive library to which fluorescently labeled probes are added to automate the detection process of the differentially expressed genes. One study using this method compared a localized versus a metastatic form of Ewing’s sarcoma and demonstrated that 90% of the genes examined had expression levels that differed between the two cancers by more than twofold⁵.

Another area of interest for future development is protein-based biochips. These biochips could be used to array protein substrates that could then be used for drug-lead screening or diagnostic tests. If a biosensor apparatus is built into these biochips a further application might be to measure the catalytic activity of various enzymes⁶. The ability to apply proteins and peptides on a wide variety of chip substrates is currently an area of intense research. The goal is to be able to control the three-dimensional patterning of these proteins on the chips through either nano-patterning on single layers or protein self assembly⁷.

The future will also see novel practical extensions of biochip applications that enable significant advances to occur without major new technology engineering. For example, a recent study described a novel practical system that allowed high-throughput genotyping of single nucleotide polymorphisms (SNPs) and detection of mutations by allele-specific extension on standard primer arrays. The assay is simple and robust enough to enable an increase in throughput of SNP typing in non-clinical as well as in clinical labs, with significant implications for areas such as pharmacogenomics⁸.

Finally, another development of protein biochips involves the use of powerful detection methodologies such as surface plasmon resonance (SPR). A recent study describes the use of SPR to detect the interaction between autoantibodies and 2-glycoprotein I (a2GPI) immobilized on protein sensor chips, this interaction being correlated with lupus. SPR enabled the interaction to be detected at a very low density of protein immobilization on the chip, and this approach therefore has significant potential for the future⁹.

Conclusions

As this fast-maturing field already boasts sales of products, biochips are likely to have a significant business future. We can expect that advances in microfluidic biochip technology will enable the miniaturization of devices that will allow highly sensitive analysis of complex biological interactions in real time. These advances promise to transform genetic diagnostics and drug screening because of their reproducibility, low cost, and speed.