Biochip: A help in Gene Therapy

Mr.Yogesh Chaudhari

In the mid-1990s, a patient with
a family history of breast cancer hears of the recent discovery of a
breast-cancer-related gene,

BRCA1,

and seeks to learn whether she
herself carries a mutation in the gene's code. A blood sample is dispatched to
a center at the forefront of genetic research, where the gene is analyzed.

Results are available in weeks. In the late 90s, another patient seeks the same
information. Her blood sample goes to a sequencing laboratory at a
university-based medical center, which already has the reagents required to
amplify
BRCA1.
Results are available in days. Early in the 21st century,
a third patient seeks the information. Results are available at a nearby clinic
and in a few minutes. The testing is performed by a biochip.

Biochip is a miniaturized devices
based on a combination of microfabrication technology and life sciences. It
employ miniaturization of biological separation and assay techniques to an
extent that multiple and complex analyses can be accomplished on a
"chip" small enough to fit the palm of the hand the so-called
"lab on a chip" or micro total analysis systems (µTAS).

Fundamentally, biochips are computer-chip
look-alikes intended to speedily, reliably, and inexpensively perform
biochemical procedures that together constitute a medical test. Through their
miniaturization and automation of laboratory procedures, they replace far
slower, more cumbersome, more expensive laboratory equipment. Their current
applications lie chiefly in the analysis of genes for defects or sequence
variations. They may soon facilitate screening of molecules for potential
usefulness as drugs. The immediate goal is to enable them to serve
point-of-care diagnosis.

First Implant of Biochip

On May 10, the human biochip era was born as
volunteers of a family in Florida  became the first people to receive the
implants. Each device, made of silicon and called a VeriChip, is a small radio
transmitter about the size of a piece of rice that is injected under a person's
skin. It transmits a unique personal ID number whenever it is within a few feet
of a special receiver unit. The purpose of implantation was if any of the
Jacobs' become ill, or are injured or impaired, their medical histories can be
accessed by professionals who can scan their microchip implants

Biochip, its Parts and Working

The current,

in use

, biochip implant
system is actually a fairly simple device. biochip implant is basically a small
(micro) computer chip, inserted under the skin, for identification purposes.
The biochip implant system consists of two components; a

transponder

and
a

reader or scanner

. The transponder is the actual biochip implant. The
biochip system is a radio frequency identification (RFID) system, using
low-frequency radio signals to communicate between the biochip and reader. The
reading range or activation range, between reader and biochip is small,
normally between 2 to12 inches.

The transponder:

The transponder is the
actual biochip implant. It is a

passive

transponder, meaning it contains
no battery or energy of it's own. In comparison, an

active

transponder
would provide it’s own energy source, normally a small battery. Because the

passive

biochip contains no battery, or nothing to wear out, it has a very long life,
up to 99 years, and no maintenance. Being

passive

, it's inactive until
the reader activates it by sending it a low-power electrical charge. The reader
"reads" or "scans" the implanted biochip and receives back
data (in this case an identification number) from the biochip. The
communication between biochip and reader is via low-frequency radio waves.

The biochip-transponder consists of four parts;

computer
microchip, antenna coil, capacitor and the glass capsule.

Computer Microchip:

The microchip stores a
unique identification number from 10 to 15 digits long. The storage capacity of
the current microchips is limited, capable of storing only a single ID number.
The unique ID number is "etched" or encoded via a laser onto the
surface of the microchip before assembly. Once the number is encoded it is
impossible to alter. The microchip also contains the electronic circuitry
necessary to transmit the ID number to the "reader".

Antenna Coil:

This is normally a simple,
coil of copper wire around a ferrite or iron core. This tiny, primitive, radio
antenna "receives and sends" signals from the reader or scanner.

Tuning Capacitor:

The capacitor stores the
small electrical charge (less than 1/1000 of a watt) sent by the reader or
scanner, which activates the transponder. This "activation" allows
the transponder to send back the ID number encoded in the computer chip.
Because "radio waves" aree same frequency as the reader.

Glass Capsule:

The glass capsule "houses" the microchip, antenna coil and capacitor. It is a small
capsule, the smallest measuring 11 mm in length and 2 mm in diameter, about the
size of an uncooked grain of rice. The capsule is made of biocompatible
material such as soda lime glass. After assembly, the capsule is hermetically
is very smooth and susceptible to movement, a material such as a polypropylene
polymer sheath is attached to one end of the capsule. This sheath provides a
compatible surface which the bodily tissue fibers bond or interconnect,
resulting in a permanent placement of the biochip.

The biochip is inserted into the subject with a
hypodermic syringe. Injection is safe and simple, comparable to common
vaccines. Anesthesia is not required nor recommended. In dogs and cats, the
biochip is usually injected behind the neck between the shoulder blades.

The reader:

The reader
consists of an "exciter" coil which creates an electromagnetic field
that, via radio signals, provides the necessary energy (less than 1/1000 of a
watt) to "excite" or "activate" the implanted biochip. The
reader also carries a receiving coil that receives the transmitted code or ID
number sent back from the "activated" implanted biochip. This all
takes place very fast, in milliseconds. The reader also contains the software
and components to decode the received code and display the result in an LCD
display. The reader can include a RS-232 port to attach a computer. The reader
and biochip can communicate through most materials, except metal

The Reader or Scaner
	Notice the ID number in the LCD display.

Types of Chips

From these beginnings, three
general types of biochips have emerged

plate-based DNA arrays

,

gel-based
DNA arrays

, and

microfluidic biochips

. The plate- and gel-based
arrays use essentially the same principles to achieve the same end. On a
substrate such as a glass plate or a porous gel, the manufacturer immobilizes
the biochip's probes, a large set of nucleic acid single strands, each carrying
a known genetic sequence. On the plate, for example, the strands are
perpendicularly embedded, one next to the other in a rectilinear matrix A test
sample is applied. It could be DNA isolated (and dissociated into single
strands) from tissue, food, soil, air, or water; its composition is unknown. At
some sites in the matrix, hybridizations occur. The biochip's readout is in
essence a list of these events. Each event establishes the identity of a short
span of code in the unknown DNA. The span's location within the DNA remains
indeterminate. The probes, however, have been selected to test for overlapping
sequences. So when the hybridization data are arranged for maximum overlap (by
computer software), the identity of the full sequence can be deduced.

Current biochips have tens of
thousands of probes, each with a length of 10 to 15 bases. Such an array is
sufficient to test an individual gene throughout all or part of its length for
single-base variations from the gene's usual sequence. In particular, individual
GeneChips detect single-base variations in the human genes

BRCA1

and

BRCA2

(breast-cancer-related),

p53

(a tumor suppressor gene mutated in many
forms of cancer), and

P450

(coding for a key liver enzyme system that
metabolizes drugs). Other GeneChips analyze the genome of HIV (human
immunodeficiency virus) for variations within the code for the viral protease
and reverse transcriptase. The intent is to help predict drug resistances of a
given patient's viral strain.

The probes in DNA-array biochips are required to
overlap along the length of the gene being examined. To detect single-base
variations, further redundancies are needed. For this purpose, the probes are
provided in sets of five, within which the probes are identical to each other
except at a single site somewhere along their length, where one probe tests for
A, a second for T, a third for G, a fourth for C, and a fifth for no nucleotide
(a single-base deletion). In essence, then, a DNA-array biochip does not
conduct actual sequencing reactions. Instead, examining a specific gene, it can
test simultaneously for myriad variations on the theme of the normal gene.

Another possible form of analysis is to probe
gene expression patterns throughout a genome. For this purpose, the probes are
selected not to cover the length of an individual gene but to have sequences
characteristic of different genes (so-called partial sequence tags). Whenever a
gene is active, its code is transcribed into single-stranded messenger RNAs,
through which the gene transmits its instructions for cellular biosynthesis of
a specific protein. Hence a cell's cytoplasm contains a variety of messengers,
depending on which genes are currently at work. The messengers' hybridizations
with one or another partial sequence tag on a biochip can serve to reveal such
patterns.

The testing may be facilitated by
representational difference analysis, which compares the results for two tissue
samples. Subtraction of one set of results from the other shows which genes are
active in one sample but less active or completely inactive in the other. The
comparison might be between normal and cancer cells, or between metastatic and
nonmetastatic cancers. In this way, the evaluation may pinpoint an abnormal
cellular process occurring chiefly or even exclusively in diseased cells. The
evaluation may thus serve not only as a diagnostic tool but also as a means to
identify therapeutic targets. The HyX Gene Discovery Modules now being
manufactured by HySeq test simultaneously for expression of 30,000 to 50,000
genes.

The microfluidic chip, is different from a DNA
array. Instead of providing nucleic acid probes, it offers channels through
which fluids can flow, typically under the impetus of an applied electric
field. It also enables the fluids to meet, typically in nanoliter quantities.
Two fluids may, for instance, converge along the short arms of a Y-shaped set
of channels. Where they meet, a reaction occurs, and the results flow down the
stem of the Y, to be sensed at the channel's far end. Alternatively, the
outcome fluid may encounter another reagent delivered at a subsequent junction
and undergo a further reaction .

The fluids can be solutions of nucleic acids,
which, being negatively charged, are highly suitable for being propelled by
electric fields. Indeed, such propulsion drives the gel electrophoresis
essential for current methods of DNA sequencing. In principle, therefore,
microfluidic technology could itself conduct DNA sequencing. It might also
conduct PCR amplification of DNA. Toward such ends, efforts are being made to
devise biochips that control not only reagent flows but also temperature (e.g.,
the cyclic heatings and coolings required for PCR). Developers envision a
biochip that can amplify DNA and determine its sequence or subject it to other
tests, start to end.

Microfluidics technology might test whether a
patient's blood contains a specific antibody or whether an antibody binds to a
specific protein. Among current types of biochips, microfluidic chips are the
only ones currently capable of such functions. However, gel-based arrays may
soon become usable for non-DNA applications. One possibility currently being
pursued is a protein array designed to analyze an enzyme's activity, or a
drug's interaction with intended (and unintended) targets, or an antibody's
specificities. Such analyses might serve not only diagnostic testing but also
drug development.

Applications of Biochips

Genomics

Genomics is the study of gene
sequences in living organisms and being able to read and interpret them. The
human genome has been the biggest project undertaken to date but there are many
research projects around the world trying to map the gene sequences of other
organisms. The use of Biochip facilitate:

• Automated genomic analysis
  including genotyping, gene expression
• DNA isolation from complex
  matrices with aim to increase recovery efficiency
• DNA amplification by optimizing
  the copy numberDNA hybridization assays to improve speed and  stringency

Proteomics

Proteome analysis or Proteomics is the investigation
of all the proteins present in a cell, tissue or organism. Proteins, which are
responsible for all biochemical work within a cell, are often the targets for
development of new drugs. The use of Biochip facilitate:

• High throughput proteomic
  analysis
• Multi-dimensional
  microseparations (pre LC/MS) to achieve high plate number
• Electrokinetic sample injection
  for fast, reproducible, samples
• Stacking or other
  preconcentration methods (as a precursor to biosensors) to improve
  detection limits
• Kinetic analysis of
  interactions between proteins to enable accurate, transport-free kinetics

Cellomics

Every living creature is made up of cells, the
basic building blocks of life.. Cells are used widely by for several
applications including study of drug cell interactions for drug discovery, as
well as in biosensing. The use of Biochip facilitate:

• Design/develop
  "lab-in-cell" platforms handling single or few cells with
  nanoprobes in carefully controlled environments.
• Cell handling, which involve
  sorting and positioning of the cells optimally using DEP, optical traps
  etc.
• Field/reagent based cell lysis,
  where the contents of the cell are expelled out by breaking the membrane,
  or increase the efficiency of transfection using reagents/field
• Intracellular processes to obtain
  high quality safety/toxicity ADME/T data

Biodiagnostics and (Nano) Biosensors

Biodiagnostics or biosensing is the field of
sensing biological molecules based on electrochemical, biochemical, optical,
luminometric methods. The use of Biochip facilitate:

Genetic/Biomarker Diagnostics, development of
Biowarfare sensors which involves optimization of the platform, reduction in
detection time and improving the signal-to-noise ratio

• Selection of detection platform
  where different formats such as lateral flow vs. microfluidics are
  compared for ease/efficiency
• Incorporation of suitable
  sensing modality by evaluating tradeoffs and downselect detection modes
  (color / luminometric, electrochemical, biochemical, optical methods) for
  specific need

Protein Chips for Diagnosis and Analysis of Diseases

The Protein chip is a micro-chip with its surface modified to detect
various disease causing proteins simultaneously in order to help find a cure for
them. Bio-chemical materials such as antibodies responding to proteins,
receptors, and nucleic acids are to be fixed to separate and analyze protein.

The Human
Genome Project, as one of the most significant scientific achievements of the
20 th century, was madepossible by the intensive use of biochip arrays capable
of decoding large stretches of human DNA sequences. Another recent high profile
use of the technology was the decoding of the SARS virus gene sequence in a
matter of months – a huge change from the years spent decoding the influenza
virus or the smallpox virus

 Apart from
resequencing and gene expression study, they may also be employed in
immunoassay  , nucleic acid amplification  , sperm selection and

invitro

fertilization 
, cell analysis  and high throughput drug screening, Preparation of nucleic
acids.

This process
involves cell separation, cell lysis, deproteinisation, etc. Now the proved
technologies for cell isolation include microfiltration-based separation and
dielectrophoretic separation (using high frequencyand non-uniform electric
field to induce the dipoles in different cells to have them separated
).Biochemical reaction. Due mainly to the limitation in detection sensitivity,
amplification of DNA derived from blood or tissue biopsies is often required.
For example, detection of a heterozygous cancer gene against a background of
thousands of "normal" genes in a tumor biopsy obviously requires
efficient and specific amplification of the target. At present, the research on
chip-based nucleic acid amplification has made some good progress.  At the
University of Pennsylvania nucleic acid amplifications such as multiplex PCR,
degenerate oligonucleotide primed (DOP)-PCR, reverse transcriptase (RT)-PCR and
ligase chain reaction (LCR) were performed using silicon-glass chips. In
addition, researchers at Perkin-Elmer used plastic chips for PCR reactions.
Fast PCR reaction has also been performed by Manz’ group at Imperial College in
London by allowing the reaction mixture flow through three different
temperature zones maintained on the glass chips.

Conventional PCR in solution can be problematic because it
is often difficult to detect low-abundance messages by gel separation and
competition between different targets, for primers means that amplification of
certain sequences is favored over others. Mosaic Technologies developed a
solid-phase PCR system in which sets of two primers (one in each orientation of
the target DNA) are arrayed onto an acrylamide film and mixed with DNA sample
and PCR reagents in solution. If the sample contains the target sequence, DNA
is synthesized from the ends of the primers and, as thermal  cycling continues,
amplified double-stranded DNA forms loops or bridges between the primers, thus
avoids the problem of competition. Another innovative approach for sample
preparation is the massively parallel solid-phase cloning, which is being
developed at Lynx Therapeutics. This method enables the simultaneous cloning of
hundreds of thousands of individual DNA fragments in a sample to be possible,
and the subsequent collection of 10 5 clones of each molecule on individual 5
micrometer beads as well.

Assays: There
are two most common approaches for on-chip detection of chemical reactants. One
is the separation-based detection method called capillary electrophoresis and
the other is the on-chip affinity-binding assay. Silicon chip-based capillary
electrophoresis was first performed by Pace at DuPont in 1983. Later on glass
chip-based capillary electrophoresis was conducted jointly by researchers at
Ciba-Geigy and the University of Alberta . DNA mutation detection and sequence
analysis were first reported by Mathie’s group at University of California at
Berkeley . This group has separated DNA fragments in less than 2 minutes. Additionally,
they has resulted in the separation of chip amplified multiplex PCR amplicons
for diagnosing Duchenne/Becker muscular dystrophy .

DNA and
protein microarrays are examples of affinity binding assay chips. DNA chips are
the most common known research platforms working through probe-target
hybridization. The complexity of the hybridization reaction depends on the
length of the immobilised DNAs as well as the application requirement (mutation
detection vs. gene expression study). There are many reports on the
applications of DNA chips for resequencing , mutation detection and expression
monitoring . For resequencing of DNA using Affymetrix’s strategy, every
nucleotide position in a gene exon or mutational hot spot has to be
interrogated, a set of four oligonucleotides is generally designed that spans
each position in the target sequence, differing only in the identity of the
central base. The relative intensity of hybridization to each series of probes
at a particular location identifies the base. Each set of oligonucleotides is
offset by one base, so that they can be arranged in order by analyzing
overlaps, a process known as tiling. In a study by Chee et al., the entire
human mitochondrial genome was resequenced by hybridization to an array that
contained approximately 135 000 oligonucleotides positioned 35 m apart . Up to
99 percent of the mitochondrial sequence could be read correctly.

Moreover, a
total of 505 polymorphisms of the mitochondrial DNA were detected from ten
African subjects. High density oligonucleotide arrays can be used to
characterize, in parallel, sequence variations of several genes in a
population. Now the companies using DNA chip for resequencing purpose include
Hyseq. Affymetrix also developed a GeneChip bioinformation system, which
bridges the chip-based resequencing work and bioinformatics. The sequencing
results can be placed into database directly for further analysis. DNA chip
could also be employed for mutation detection. For example, Hacia et al.
detected 24 heterozygous mutations located in exons of the

BRC

A1 gene
using DNA chip attached with 96 000 oligonucleotide probes .

Another use
of DNA chip is for gene expression study. Using DNA chip for gene expression
analysis brings the tremendous strike for disease diagnosis and drug screening.
Lockhart et al. quantitatively monitored 21 messenger RNA targets from the
entire RNA populations of a mouse T-cell line using an array of 65 000
different 20-mer oligonucleotide probes, which were designed to hybridize with
a total of 114 known mouse genes . Changes in expression were detected for 20
other mRNAs after the induction of cytokine production, RNAs present at a
frequency higher than 1 300 000 were detected, and the amounts of mRNA could be
quantified up to a frequency of 1 300. DeRisi et al. used a high density
microarray of 1 161 DNA elements to search for differences in gene expression
associated with tumour suppression in a human melanoma cell line . By comparing
different fluorescence signals representing hybridization to each arrayed gene,
they determined the relative abundance in the two samples of mRNAs
corresponding to each gene and concluded that the tumorigenic properties can be
suppressed by introduction of a normal human chromosome.

For DNA chip makers and users,
further effort is needed to have the hybridization process optimized. The
hybridization interaction taking place on a DNA chip is no longer a
solution-phase  reaction but a solid-liquid one. Hence, the hybridization
process may take much longer time and the DNA secondary structure issue may
also hinder the hybridization on the chip. The conditions that favor good
duplex formation on the chip are also likely to promote intrastrand duplex
formation, or the formation of tertiary structures in DNA in the sample. To
solve these problems, researchers developed probes made of peptide nucleic
acids (PNA) to address the intrastrand hybridization. For DNA-DNA
hybridization, magnesium ions are needed to counteract the interstrand
repulsion between the negatively charged sugar phosphate backbones.
Single-stranded DNA thus assumes ‘native’conformations including complex
secondary and tertiary structures. PNA-DNA hybrid formation, on the other hand,
does not require salts. Therefore, the DNA strands are not folded and the target
sequences are accessible. Because the duplex stability of PNA-DNA is higher
than that of DNA-DNA, binding is more specific single-base mismatches are more
readily detectable. To enable DNA to be enriched on the chip is one way to
accelerate the parallel hybridization speed. For example, Nanogen developed
active electronic biochips for the analyses of DNA/RNA sequences. Using this
type of active chip, DNA targets can be enriched very quickly in proximity to
the surface of the chip and the hybridization reaction can thus be made much
quicker than usual reducing the time required from hours to seconds.

Data
acquisition

: At present, the most common detection method applied to DNA
chips is fluorescent detection due to its high reproducibility and sensitivity.
Sensitivity and quantitative dynamic range of the fluorescent reporter is
important, especially in mutation detection, where heterozygotes may be under
investigation. A reporter must have a relative discrimination ratio
significantly better than 2:1 for the wild type compared with the mutant
sequence in order to facilitate the detection of a heterozygote. Most present
systems for diagnostic applications have discrimination ratios of around 5:1.
Also, there are many other methods under development, such as the
time-of-flight mass spectrometry (TOF-MS), the methods using
chemiluminescence/luminescence, optical fibers, diode array detectors, direct
electrical charge change detection, and piezoelectric readout. For example,
with the technology developed at Sequenom, the probes are bound to the chip
surface through a photocleavable linker. After hybridization, a laser is used
to cleave the attached molecule and the bound oligonucleotides at each location
are “incised”. The released molecules were then detected by a time-of-flight
mass spectrometer. Though only short DNA sequences can be analysed by mass
spectrometry at present, the longer ones may be possible by given more time.
Lloyd Smith’s group at the University of Wisconsin used PNA probes and TOF-MS
to analyze polymorphic sites within the human tyrosinase gene. Whatever the
reporter system of choice, some sophisticated gadgetry and software are needed
to interpret the readout into meaningful data. Some companies, such as General
Scanning and Molecular Dynamics, have launched their scanning confocal
instruments which may detect thousands of probing results on the chip surface
at micrometer resolution.

Apart from
the microarrays mentioned above, researchers are developing many other
microchips with different microstructures (e.g. microchannel, micro reaction
chamber, microfilter) and functions. Some typical examples include capillary
electrophoresis chips, and cell sorting chips, immunoassay chips, and nucleic
acid amplification chips. All of these will lay a solid foundation for the
departure in the development of laboratory-on-a-chip and miniaturization of
analytical instruments.

Integration of
Functional Genomics and Biochip Technology for Drug Discovery and Diagnostic

Current drug
discovery pipeline relies upon two critical aspects - combinatorial chemical
library and high-throughput screening. The output of the pipeline is limited by
the availability of gene targets for screening, and the diversity of the
compounds to be screened. By integrating state-of-the-art technologies such as
biochips, cell-based assays and bioinformatics have established a functional
genomics platform for disease gene identification and drug discovery.Technology
platform performs comprehensive biochip-based gene expression profiling and cell
function profiling to identify disease pathways and genes that are targeted by
effective drugs. The combination of these technologies will greatly accelerate
the identification of disease-related genes as potential drug targets, the
understanding of action mechanisms and toxicology of drugs.

 Implantable Microchips for Drug Delivery

There is growing
interest in the use of microchips as delivery systems for pharmaceuticals,
diagnostic reagents, and other chemicals.Prototype microchips contain an array
of sealed micro-reservoirs, each of which is filled with a chemical to be
delivered.The technology has advanced to allow controlled chemical release from
subcutaneously implanted microchip devices. This in vivo demonstration of
chemical release from a microchip represents a significant step toward the
development of "smart" drug delivery systems.

Biosensors
Encapsulating Human Gene Functions: Applications in Drug Discovery and
Diagnostics

Human receptors
that are important therapeutic and diagnostic targets are complexes of many
proteins. Ligand dependent function for most of the receptors cannot be
established in vitro due to complexity and lack of ligand mediated sensitivity
and selectivity. To circumvent this problem ligand dependent functions for many
of the human nuclear receptors have been established in yeast. These highly
sensitive LiveSensors are robust and amenable to miniaturization on chip-based
format.

Microelectronic Array Devices and Systems
for DNA Diagnostic, Pharmacogenomic and Drug Discovery Applications-

Active
microelectronic array systems are being developed for applications in DNA
diagnostics, pharmacogenomic research and drug discovery. These microarray
devices allow charged reagent and analyte molecules, including DNA, RNA,
oligonucleotide probes, amplicons antibodies, proteins, enzymes,
nanostructures, cells, and even semiconductor structures to be moved to or from
any of the microscopic test sites on the device surface. A research laboratory
system has been designed to provide the end-user with "make your own
chip" capabilities. Applications include analysis, infectious disease
diagnostics, gene expression analysis, and ultimately on-chip amplification
capabilities. This system allows assays to be carried out rapidly and with high
selectivity. Nanogen’s electronic hybridization technology has been shown to
provide highly reliable results for "problematic" genotyping. Nanogen
is now also developing other versions of its electronic hybridization
technology for general laboratory and high throughput applications.
Additionally, in collaboration with Aventis/Selectide R&D groups, Nanogen
is investigating the use of active electronic devices for the development of
high throughput screening systems for carrying out kinase, phosphatase, and protease
enzyme inhibitor assays. This technology may prove useful for drug discovery
applications.

Biochip and
Biosensor Technology Development

Bioelectronic
Detection of DNA Hybridization:

 A whole new
class of DNA diagnostic devices is being enabled by the development of
non-flourescence detection techniques. Three approaches are being developed and
compared. The first is label-free impedimetric detection of DNA hybridization
using microlithographically fabricated arrays of interdigitated electrodes on
oxidized silicon. In a second approach, this detection is enhanced by the use
of colloidal gold nanoparticles that serve as labels of target DNA. The third
approach uses an electroactive layer of inherently conductive polypyrrole to
provide for covalent attachment of DNA probes as well as provide enhanced redox
detection sensitivity to labels such as ferrocene.

Microarrays of
Gel Immobilized Compounds: Massive Parallel Analyses of Specific Interactions
and Chemical and Enzymatic  Reactions  

MicroArrays of
Gel Immobilized Compounds on a Chip (MAGIChips) are produced by immobilizing
oligonucleotides, DNA, enzymes, antibodies, and other compounds on a
photopolymerized micromatrix of polyacrylamide gel pads 100x100x20 µm and
smaller in size. Alternatively, allyl-modified compounds can be embedded within
polyacrylamide gel pads by copolymerization. MAGIChips have been shown to be
efficient for analysis of nucleic acid hybridization, specific binding of DNA
with proteins, and low-molecular-weight compounds, and protein-protein
interactions. The three-dimensional gel pads of the microarrays can be used as
nanoliter-sized microtest tubes to perform ligation, single-base extension, PCR
amplification of DNA, and other reactions. The fluorescence microscope has been
devised for analyzing reactions on MAGIChips, including quantitative and
real-time monitoring of hybridization, measuring the thermodynamic parameters
of DNA duplexes containing different modified nucleotides, and measuring the
kinetics of enzymatic reaction. On-chip MALDI-TOF mass spectrometry was
successfully tested for analysis of DNA and protein interactions. Application
of these biochips for detection of human polymorphism and mutations,
identification of microorganisms and their drug resistant and toxin-bearing
strains, and in different fields of biotechnology and medicine will be
described.

Color-Coded Clustered Image Maps (CIMs)
for Gene Expression Fingerprinting and Cancer Drug Discovery

It has proved
easier to use gene expression profiles for classification of tumors and for
prognosis than it has been to integrate such profiles into the drug discovery
process. We and our collaborators have used both cDNA microarrays and
oligonucleotide chips to characterize patterns of gene expression in 60 human cancer
cell lines used by the National Cancer Institutes drug discovery program. Since
those cells have also been characterized by their patterns of sensitivity to
>70,000 chemical agents, we are afforded a unique opportunity to relate the
genomics to the pharmacology.

Hetero-Functional
Biochip Platform for Applications in Proteomics and Genomics

The biochip is a self-contained device which allows simultaneous detection of
various types of biotargets using different bioreceptors (e.g., antibodies,
nucleic acids, enzymes, cellular probes) on a single system. The biochip sensor
array device, which is based on an integrated circuit (IC), is designed using
complementary metal oxide silicon (CMOS) technology and includes photosensors,
amplifiers, discriminators and logic circuitry on board. The highly integrated
biochip is produced using the capability of fabricating multiple optical
sensing elements and microelectronics for up to 100 sensing channels on a
single IC. The capability of large-scale production using low-cost IC
technology is an important advantage. The assembly process of various
components is made simple by cost-effective integration of multiple elements on
a single chip. The usefulness and potential of the biochip technology for
proteomics and genomics applications will be discussed. Rapid, simple,
cost-effective medical devices for screening multiple medical diseases and
infectious pathogens are essential for early diagnosis and improved treatments
of many illnesses. An important factor in medical diagnostics is rapid,
selective, and sensitive detection of biochemical substances (proteins,
metabolites, nucleic acids), biological species or living systems (bacteria,
virus or related components) at ultratrace levels in biological samples (e.g.,
tissues, blood and other bodily fluids). The performance of the biochip device
is illustrated with fluorescence detection of both DNA probes specific to gene
fragments of the Mycobacterium Tuberculosis (TB) and the human
immuno-deficiency virus (HIV) systems, and of antibody probes targeted to the
cancer suppressor gene .

A
View of the Future

The immediate
prospects for biochip technology depend on a range of technologic and economic
issues. One is the question of chip reusability. Current biochips are of necessity
disposable, in part because the current devices are not physically robust. For
example, nucleic acid probes tend to break away from a supporting glass plate.
A decade from now, this problem may have been better addressed, making the
chips more reusable, and perhaps at the same time permitting probes with longer
spans of genetic data than are feasible today. In this way, a manufacturing
improvement might facilitate more powerful forms of genetic analysis. On the
other hand, it may be better to manufacture biochips so inexpensive that they
can be used once and then discarded. Another issue is biochip versatility.
Current biochips are single-purpose, hardwired devices. Even if future biochips
do not become programmable, in the fashion of computer chips, they may become
usable for multiple purposes, such as the analysis of a tissue sample for
numerous pathogens.

An overarching
issue is standardization. For diagnostic purposes, any medical test should be
administered, and its results interpreted, in a standardized way. Beyond that,
it seems desirable for biochips performing different tests to have an output
detectable by the same readout device. Hence, a race is underway to create a
biochip platform or motherboard capable of handling a wide range of biochips,
irrespective of the internal details of a given chip's function. In particular,
two companies, Affymetrix and Molecular Dynamics, have formed the Genetic
Analysis Technology Consortium, or GATC (a name that also represents the four
nucleotides that carry genetic code in DNA). The hope is to establish
industry-wide standards for the reading of biochips. 

An optimum
biochip strategy is not yet discernible. Even as efforts at standardization
proceed, novel technologic options continue to emerge. In one idea, called
optical mapping, DNA is elongated and fixed on glass to ensure that its full
extent is accessible to reagents or laser light, and that its sequence data
retain their order, even after enzymatic cleavages. Another idea is that
standard compact disk (CD) technology, as currently used for storage, writing,
and readout of audio, video, and computer data, might be adapted to carry the
probes or other reagents held on a biochip, and that a CD player might be
adapted to read test results. After all, a standard CD player obtains its data
by directing a laser beam at a succession of microscopic holes. The holes might
be redesigned to house molecules that would interact with laser light in
characteristic ways, thereby providing a test result. In the face of continued
innovation, biochip developers must deal with the danger of being locked
prematurely into a technologic choice.

For now, it is
expected that biochips much like those already available for the analysis of
human genes such as

BRCA1, BRCA2, p53,

and

P450

will soon find
use for other tests. Certainly, the public availability of increasing
quantities of sequence data for various loci within the human genome's three
billion bases has been making it easier to analyze an increasing number of
genes with variant versions (alleles) known to be linked to disease. Thus, the
list of expected near-term biochip applications includes chips pertinent to
thalassemias, Tay-Sachs disease, spinal muscular atrophies, retinoblastomas,
neurofibromatosis, various dystrophies, amyotrophic lateral sclerosis,
Huntington's disease, Gaucher's disease, fragile X syndrome, hemophilias,
cystic fibrosis, and various polyposes and enzyme deficiencies. Expected
biochips will also test for a variety of pathogens.

Meanwhile, the
types of testing performed by biochips can be expected to expand. The test
results may, for instance, become quantitative, so that biochips address not
only such questions as whether a gene occurs in a DNA sample or is being
expressed in a cell but also how much it is being expressed. Many strategies
for such testing are already being explored. One example is a
competitive-binding assay in which a ligand in a test sample competes with a
fluorescently labeled substance for binding to a reagent. The amount of fluorescence
measured subsequently in the reagent is then an inverse measurement of how much
ligand the test sample contained.

The more crucial
problem lies in giving quantitative data a useful clinical interpretation.
Informed of a particular level of gene expression, a physician would want to
know what the implications are for disease progression. The physician would
also want to know whether the findings predict a patient's likelihood of
responding to one or another intervention. Such issues are entirely independent
of any technologic challenges arising in developing the pertinent biochips. It
is hoped that through their acceleration of the acquisition of data, biochips
will assist in correlating genetic variation, gene expression, and protein
levels with a patient's clinical status, prognosis, and responsiveness to
treatment. The goal is to discover how to tailor medical care to a patient's
genetic makeup, an endeavor now being called pharmacogenomics. Its beginnings
can be seen in exploration of genes such as

p53

and

P450.

Pharmacogenomics constitutes one of the most intensive emerging areas in
pharmaceutical research.

Similar to
the nanometer-scale fabrication demanded by the microelectronic industry,
nano-sized biochips are also desired for some applications but not all.
Researchers found that the counterparts of machine components can be found
among natural molecules and biological assemblies of molecules, for example,
collagen is a cable, an antibody is a clamp, DNA is a memory device, and
membrane proteins are pumps. Currently there are no nano-sized biochips having
been on reported; however, some basic parts, such as 0.5-mm diameter, 30-mm
long lipid tubules, 0.7- to 0.8-mm diameter cyclic peptide nanotubes, and the
metallocene molecular gear, have been achieved using molecular sself
assembling  technology. The design and synthesis of molecules that mimic
mechanical devices provide the grounds for some optimism for the future
nano-sized biochip development. The ultimate goal of any biochip development effort
is to build a fully integrated analytical system a “laboratory-on-a-chip”.
Considerable strides have been made in this type of integration. Chip with
heaters, valves, pumps, microfluidic controllers, and eletrochemical and
electroluminescent detectors have been produced at Nanogen, Affymetrix,
University of Pennsylvania, Lawrence Livermore National Laboratory and
University of Michigan. Researchers at Nanogen made the first report on
laboratory-on-a-chip. Using a technique called dielectrophoresis they separated
bacterial cell from human blood cells on the bioelectronic chips. The isolated
bacterial cells were then lysed by applying a series of high-voltage pulses.
The released DNA/RNA was further examined through electronic hybridization
process. Using this type of chip-based shrinking, laboratory hour-long

conventional
bioassays may be finished in minutes. Another important application of biochips
lies in high throughput or ultra-high throughput screening of drugs. This
requires the effective integration of biochip technology, robotic automation
technology, combinatorial chemistry and receptor binding assay. Using biochip
technology to analyze and screen natural compounds from plants is vital for the
development of the traditional Chinese   medicine. The combinative-use of
biochip technology, micro-liquid dispensing technology and diversified
detection technologies may bring forth the breakthrough for new drug
discovery  

Conclusion:

Biochips are fast, accurate, miniaturized, and can be
expected to become economically advantageous--attributes that make them
analogous to a computer chip. One expects to see an accelerated trend of
ultraminiaturization, perhaps involving entirely novel media, and an increased
ability to analyze not only genetic material but also other types of biologic
molecules. One expects, too, an eventual harmonization of technologies, so that
dominant fabrication strategies will emerge, at least for certain types of
applications, including a favored format for genetic analysis and another for
antibodies and other proteins. Since the potential applications are vast, both
for research and for clinical use, the potential markets for biochips will be
huge, a powerful driving force for their continued development

The rapid
advancement in biochip research has already become the attention focus of both
academia and industry. After restless efforts over a decade this technology is
now gradually becoming matured. It is estimated that biochip industry may
eventually generate a market of over 10 billion US dollars in the 21st century.
The great potential it has may bring a revolution to many fields in life
science and medicine in the coming century. In the beginning of this year
Nature Genetics magazine launched a supplement issue dedicated to the microarray
technology one of the most widely used approach for DNA and protein analysis.
Several important applications have been described and discussed in this issue,
topics include DNA resequencing and mutation detection, gene expression study,
bioinformatics, population genetics, discovery of new drugs, etc. This is the
first time that an entire issue was set up for one topic, which demonstrates
how important this field is. Microarrayed chips account for only one type of
biochips used for affinity binding assays. There are many other types of chips
existing along with microarrays. One can imagine how significant these
chips’roles would be  once they were integrated for our diversified use. In 5
10 years, portable laboratories built using chips may be commercially available
like the notebook computer we see today. By then we may afford our
individualized analyzer accompanying us to anywhere in the world, and our life
quality may be very different from now.

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About Authors

Mr.Yogesh Chaudhari, Baheti Akshay M., Mrs.S.P.Ingale, Dr.S.R.Parakh

Mr.Yogesh Chaudhari

MAEER’s Maharashtra Institue of Pharmacy, Pune-411038

Baheti Akshay M.

Asst professor in Pharmacognosy, MAEER’s Maharashtra Institute of Pharmacy, Pune 411038

Mrs.S.P.Ingale

Lecturer in Pharmacology, MAEER’s Maharashtra Institute of Pharmacy, Pune 411038

Dr.S.R.Parakh

Principal, MAEER’s Maharashtra Institute of Pharmacy, Pune 411038