The development of
biochips is a major thrust of the rapidly growing
biotechnologyBiotechnology is technology based on biology, agriculture, food science, and medicine. Modern use of the term usually refers to genetic engineering as well as cell- and tissue culture technologies...
industry, which encompasses a very diverse range of
research efforts including
genomicsGenomics is the study of the genomes of organisms. The field includes intensive efforts to determine the entire DNA sequence of organisms and fine-scale genetic mapping efforts. The field also includes studies of intragenomic phenomena such as heterosis, epistasis, pleiotropy and other...
,
proteomicsProteomics is the large-scale study of proteins, particularly their structures and functions. Proteins are vital parts of living organisms, as they are the main components of the physiological metabolic pathways of cells. The term "proteomics" was first coined in 1997 to make an analogy with...
, and pharmaceuticals, among other activities. Advances in
these areas are giving scientists new methods for unravelling the complex
biochemical processes occurring inside cells, with the larger goal of
understanding and treating human diseases. At the same time, the
semiconductor industryThe semiconductor industry is the aggregate collection of companies engaged in the design and fabrication of semiconductor devices. It formed around 1960, once the fabrication of semiconductors became a viable business...
has been steadily perfecting the science of
micro-miniaturization. The merging of these two fields in recent years has
enabled biotechnologists to begin packing their traditionally bulky
sensing tools into smaller and smaller spaces, onto so-called biochips. These
chips are essentially miniaturized laboratories that can perform hundreds or
thousands of simultaneous biochemical reactions. Biochips enable researchers
to quickly screen large numbers of biological analytes for a variety of
purposes, from disease diagnosis to detection of bioterrorism agents.
Definition
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 output and speed. Biochips can also be used to perform techniques such as
electrophoresisElectrophoresis is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. This electrokinetic phenomenon was observed for the first time in 1807 by Reuss , who noticed that the application of a constant electric field caused clay particles...
or PCR using
microfluidicsMicrofluidics deals with the behavior, precise control and manipulation of fluids that are geometrically constrained to a small, typically sub-millimeter, scale.Typically, micro means one of the following features:* small volumes...
technology (Fan, 2009; Cady, 2009).
History
The development of biochips has a long history, starting with early work on
the underlying
sensorA sensor is a device that measures a physical quantity and converts it into a signal which can be read by an observer or by an instrument. For example, a mercury-in-glass thermometer converts the measured temperature into expansion and contraction of a liquid which can be read on a calibrated glass...
technology. One of the first portable, chemistry-based
sensors was the
glass pH electrodeA glass electrode is a type of ion-selective electrode made of a doped glass membrane that is sensitive to a specific ion.- Types :Almost all commercial electrodes responds to single charged ions, like H+, Na+, Ag+. The most common glass electrode is the...
, invented in 1922 by
Hughes (Hughes, 1922). Measurement of
pHpH is a measure of the acidity or basicity of a solution. It is defined as the cologarithm of the activity of dissolved hydrogen ions . Hydrogen ion activity coefficients cannot be measured experimentally, so they are based on theoretical calculations...
was accomplished by
detecting the potential difference developed across a thin glass membrane
selective to the permeation of hydrogen ions; this selectivity was achieved
by exchanges between H
+ and SiO sites in the glass. The basic concept of
using exchange sites to create permselective membranes was used to develop
other
ion sensorsAn Ion-selective electrode is a transducer which converts the activity of a specific ion dissolved in a solution into an electrical potential which can be measured by a voltmeter or pH meter. The voltage is theoretically dependent on the logarithm of the ionic activity, according to the Nernst...
in subsequent years. For example, a K
+ sensor was
produced by incorporating
valinomycinValinomycin is a dodecadepsipeptide, that is, it is made of twelve alternating amino acids and esters to form a macrocyclic molecule.Valinomycin is obtained from the cells of several Streptomyces strains, one of them "S. tsusimaensis"....
into a thin membrane (Schultz, 1996).
Over thirty years elapsed before the first true
biosensorA biosensor is a device for the detection of an analyte that combines a biological component with a physicochemical detector component.It consists of 3 parts:* the sensitive biological element A biosensor is a device for the detection of an analyte that combines a biological component with a...
(
i.e. a
sensor utilizing biological molecules) emerged. In 1956, Leland Clark
published a paper on an
oxygen sensing electrodeThe Clark electrode is an electrode that measures oxygen on a catalytic platinum surface using the net reaction:- History :Leland Clark had developed the first bubble oxygenator for use in cardiac surgery...
(Clark, 1956_41).
This device became the basis for a
glucoseGlucose , a monosaccharide also known as - grape sugar, blood sugar, or corn sugar, is a very important carbohydrate in biology. The living cell uses it as a source of energy and metabolic intermediate...
sensor developed in 1962 by Clark
and colleague Lyons which utilized
glucose oxidaseThe glucose oxidase enzyme binds to beta-D-glucopyranose and aids in breaking the sugar down into its metabolites...
molecules embedded in a
dialysisIn medicine, dialysis is primarily used to provide an artificial replacement for lost kidney function due to renal failure...
membrane (Clark, 1962). The
enzymeEnzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process are called substrates, and the enzyme converts them into different molecules, called the products. Almost all processes in a biological cell need enzymes to occur at...
functioned in the
presence of glucose to decrease the amount of oxygen available to the oxygen electrode, thereby relating oxygen levels to glucose concentration. This and
similar biosensors became known as enzyme electrodes, and are still in use
today.
In 1953,
Watson and CrickWatson and Crick refers to the duo of James D. Watson and Francis Crick who, using x-ray data collected by Rosalind Franklin, proposed the double helix structure of the DNA molecule in 1953...
announced their discovery of the now familiar
double helixIn geometry a double helix typically consists of two congruent helices with the same axis, differing by a translation along the axis, which may or may not be half-way....
structure of
DNADeoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms and some viruses. The main role of DNA molecules is the long-term storage of information...
molecules and set the stage for
geneticsGenetics, , a discipline of biology, is the science of heredity and variation in living organisms. The fact that living things inherit traits from their parents has been used since prehistoric times to improve crop plants and animals through selective breeding...
research that continues to the present day (Nelson, 2000). The development
of
sequencingIn genetics and biochemistry, sequencing means to determine the primary structure of an unbranched biopolymer. Sequencing results in a symbolic linear depiction known as a sequence which succinctly summarizes much of the atomic-level structure of the sequenced molecule.-DNA sequencing:DNA...
techniques in 1977 by
GilbertWalter Gilbert is an American physicist, biochemist, molecular biology pioneer, and Nobel laureate.-Biography:Gilbert was born in Boston, Massachusetts into a Jewish family and educated at the Sidwell Friends School, Harvard University and the University of Cambridge, later joining the faculty at...
(Maxam, 1977) and
SangerFrederick Sanger, OM, CH, CBE, FRS is an English biochemist and twice a Nobel laureate in chemistry. He is the fourth person to have been awarded two Nobel Prizes.- Early years :...
(Sanger, 1977) (working separately) enabled researchers to
directly read the genetic codes that provide instructions for
protein synthesisProtein synthesis is the process in which cells build proteins. The term is sometimes used to refer only to protein translation but more often it refers to a multi-step process, beginning with amino acid synthesis and transcription of nuclear DNA into messenger RNA which is then used as input to...
. This research showed how hybridization of complementary single
oligonucleotideAn oligonucleotide is a short nucleic acid polymer, typically with twenty or fewer bases. Although they can be formed by bond cleavage of longer segments, they are now more commonly synthesized by polymerizing individual nucleotide precursors...
strands could be used as a basis for DNA sensing. Two
additional developments enabled the technology used in modern DNA-based
biosensors. First, in 1983
Kary MullisKary Banks Mullis is an American biochemist and Nobel laureate.Mullis shared the 1993 Nobel Prize in Chemistry with Michael Smith. Mullis received the prize for his development of the Polymerase Chain Reaction , a process first described by Kjell Kleppe and 1968 Nobel laureate H. Gobind Khorana...
invented the
polymerase chain reactionIn molecular biology, the polymerase chain reaction is a technique to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence...
(PCR) technique (Nelson, 2000), a method for amplifying DNA concentrations.
This discovery made possible the detection of extremely small quantities of
DNA in samples. Second, in 1986 Hood and co-workers devised a method to label
DNA molecules with
fluorescent tagIn molecular biology and biotechnology, a fluorescent tag is a part of a molecule that researchers have attached chemically to aid in detection of the molecule to which it has been attached. The tag is some kind of fluorescent molecule . Ethidium bromide, Fluorescein and Green fluorescent protein...
s instead of
radiolabels (Smith, 1986), thus enabling hybridization experiments to
be observed optically.
The rapid technological advances of the
biochemistryBiochemistry is the study of the chemical processes in living organisms. It deals with the structure and function of cellular components such as proteins, carbohydrates, lipids, nucleic acids and other biomolecules....
and
semiconductorA semiconductor is a material that has an electrical resistivity between that of a conductor and an insulator, that is, generally in the range 10
3 Siemens/cm to 10
−8 S/cm. Devices made from semiconductor materials are the foundation of modern electronics, including radio,...
fields
in the 1980s led to the large scale development of biochips in the 1990s.
At this time, it became clear that biochips were largely a "platform"
technology which consisted of several separate, yet integrated components.
Figure 1 shows the make up of a typical biochip platform.
The actual sensing component (or "chip") is just one piece of a complete
analysis system.
TransductionA transducer is a device, electrical, electronic, electro-mechanical, electromagnetic, photonic, or photovoltaic, that converts one type of energy or physical attribute to another for various purposes including measurement or information transfer .There are two kinds of transducers...
must be done to translate the actual sensing
event (DNA binding,
oxidation/reductionRedox describes all chemical reactions in which atoms have their oxidation number changed....
,
etc.) into a format
understandable by a computer (
voltageVoltage is commonly used as a short name for electrical potential difference. Its corresponding SI unit is the volt...
, light intensity, mass,
etc.),
which then enables additional analysis and processing to produce a final,
human-readableA human-readable medium or human-readable format is a representation of data or information that can be naturally read by humans.In computing, human-readable data is often encoded as ASCII or Unicode text, rather than presented in a binary representation...
output. The multiple technologies needed to make a successful
biochip — from sensing chemistry, to
microarrayA microarray is a multiplex lab-on-a-chip. It is a 2D array on a solid substrate that assays large amounts of biological material using high-throughput screening methods.Types of microarrays include:...
ing, to signal processing —
require a true multidisciplinary approach, making the barrier to entry steep.
One of the first commercial biochips was introduced by
AffymetrixAffymetrix is a manufacturer of DNA microarrays, based in Santa Clara, California, United States. The company was co-founded by Dr. Stephen Fodor in 1992. The company was begun as a unit in Affymax N.V...
. Their
"GeneChip" products contain thousands of individual DNA sensors for use in
sensing defects, or single nucleotide polymorphisms (SNPs), in genes such as
p53p53 , is a transcription factor which in humans is encoded by the TP53 gene. p53 is important in multicellular organisms, where it regulates the cell cycle and thus functions as a tumor suppressor that is involved in preventing cancer...
(a tumor suppressor) and
BRCA1BRCA1 is a human gene, some mutations of which are associated with a significant increase in the risk of breast cancer, as well as other cancers. BRCA1 belongs to a class of genes known as tumor suppressors, which maintains genomic integrity to prevent dangerous genetic changes...
and
BRCA2BRCA2 is a protein that in humans is encoded by the BRCA2 gene. BRCA2 belongs to the tumor suppressor gene family and the protein encoded by this gene is involved in the repair of chromosomal damage with an important role in the error-free repair of DNA double strand breaks.The BRCA2 gene is...
(related to breast
cancer) (Cheng, 2001). The chips are produced using microlithography
techniques traditionally used to fabricate integrated circuits (see below).
frame
Today, a large variety of biochip technologies are either in development or
being commercialized. Numerous advancements continue to be made in sensing
research that enable new platforms to be developed for new applications.
Cancer diagnosis through DNA typing is just one market opportunity. A variety
of industries currently desire the ability to simultaneously screen for a
wide range of chemical and biological agents, with purposes ranging from
testing public water systems for disease agents to screening airline cargo
for explosives. Pharmaceutical companies wish to combinatorially screen drug
candidates against target enzymes. To achieve these ends,
DNADeoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms and some viruses. The main role of DNA molecules is the long-term storage of information...
,
RNARibonucleic acid is a biologically important type of molecule that consists of a long chain of nucleotide units. Each nucleotide consists of a nitrogenous base, a ribose sugar, and a phosphate...
, proteins,
and even living cells are being employed as sensing mediators on biochips (Potera, 2008).
Numerous transduction methods can be employed including
surface plasmon resonanceThe excitation of surface plasmons by light is denoted as a surface plasmon resonance for planar surfaces or localized surface plasmon resonance for nanometer-sized metallic structures....
,
fluorescenceFluorescence is the emission of visible light by a substance that has absorbed light of a differing, usually invisible, wavelength. Absorption of a photon triggers the emission of a photon with a longer wavelength. A shorter wavelength emission is sometimes observed from multiple photon absorption...
, and chemiluminescence. The particular sensing and
transduction techniques chosen depend on factors such as price, sensitivity, and reusability.
Microarray fabrication
The microarray — the dense, two-dimensional grid of biosensors — is the critical component of a biochip platform. Typically, the sensors are deposited on a flat substrate, which may either be passive (
e.g. silicon or glass) or active, the latter
consisting of integrated electronics or
micromechanicalMicrotechnology is technology with features near one micrometre .In the 1960s, scientists learned that by arraying large numbers of microscopic transistors on a single chip, microelectronic circuits could be built that dramatically improved performance, functionality, and reliability, all while...
devices that perform or assist signal transduction. Surface chemistry is used to
covalently bindA covalent bond is a form of chemical bonding that is characterized by the sharing of pairs of electrons between atoms, or between atoms and other covalent bonds...
the sensor molecules to the substrate medium. The fabrication of microarrays is non-trivial and is a major economic and technological hurdle that may
ultimately decide the success of future biochip platforms. The primary manufacturing challenge is the process of placing each sensor at a specific position (typically on a Cartesian grid) on the substrate. Various means exist to achieve the placement, but typically robotic micro-pipetting (Schena, 1995) or micro-printing (MacBeath, 1999) systems are used to place tiny spots of sensor material on the chip surface. Because each sensor is unique, only a few spots can be placed at a time. The low-throughput nature of this
process results in high manufacturing costs.
Fodor and colleagues developed a unique fabrication process (later used by
AffymetrixAffymetrix is a manufacturer of DNA microarrays, based in Santa Clara, California, United States. The company was co-founded by Dr. Stephen Fodor in 1992. The company was begun as a unit in Affymax N.V...
) in which a series of microlithography steps is used to
combinatorially synthesizeCombinatorial chemistry involves the rapid synthesis or the computer simulation of a large number of different but structurally related molecules or materials.-Introduction:...
hundreds of thousands of unique, single-stranded
DNA sensors on a substrate one
nucleotideNucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides play central roles in metabolism...
at a
time (Fodor, 1991; Pease, 1994). One lithography step is needed per base type; thus, a total
of four steps is required per nucleotide level. Although this technique is
very powerful in that many sensors can be created simultaneously, it is
currently only feasible for creating short DNA strands (15–25 nucleotides).
Reliability and cost factors limit the number of photolithography steps that
can be done. Furthermore, light-directed combinatorial synthesis techniques
are not currently possible for proteins or other sensing molecules.
As noted above, most microarrays consist of a Cartesian grid of sensors. This
approach is used chiefly to map or "encode" the coordinate of each sensor
to its function. Sensors in these arrays typically use a universal signalling
technique (
e.g. fluorescence), thus making coordinates their only
identifying feature. These arrays must be made using a serial process
(
i.e. requiring multiple, sequential steps) to ensure that each sensor
is placed at the correct position.
"Random" fabrication, in which the sensors are placed at arbitrary
positions on the chip, is an alternative to the serial method. The tedious and expensive positioning process is
not required, enabling the use of parallelized self-assembly techniques. In
this approach, large batches of identical sensors can be produced; sensors
from each batch are then combined and assembled into an array. A
non-coordinate based encoding scheme must be used to identify each sensor. As
the figure shows, such a design was first demonstrated (and later
commercialized by Illumina) using functionalized beads placed randomly in the
wells of an etched fiber optic
cable (Steemers, 2000; Michael, 1998) Each bead was uniquely
encoded with a fluorescent signature. However, this encoding scheme is
limited in the number of unique dye combinations that can be used and
successfully differentiated.
Protein biochip array and other microarray technologies
MicroarrayA microarray is a multiplex lab-on-a-chip. It is a 2D array on a solid substrate that assays large amounts of biological material using high-throughput screening methods.Types of microarrays include:...
s are not limited to
DNADeoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms and some viruses. The main role of DNA molecules is the long-term storage of information...
analysis;
protein microarrayA protein microarray, sometimes referred to as a protein binding microarray,provides a multiplex approach to identify protein-protein interactions, to identify the substrates of protein kinases, to identify transcription factor protein-activation, or to identify the targets of biologically active...
s,
antibody microarrayAn antibody microarray is a specific form of protein microarrays, a collection of capture antibodies are spotted and fixed on a solid surface, such as glass, plastic and silicon chip for the purpose of detecting antigens...
,
chemical compound microarrayA chemical compound microarray is a collection of organic chemical compounds spotted on a solid surface, such as glass and plastic. This microarray format is very similar to DNA microarray, protein microarray and antibody microarray...
can also be produced using biochips.
Randox Laboratories Ltd. launched Evidence, the first protein Biochip Array Technology analyzer in 2003. In protein Biochip Array Technology, the biochip replaces the
ELISAEnzyme-linked immunosorbent assay, also called ELISA, enzyme immunoassay or EIA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality...
plate or
cuvetteA cuvette is a small tube of circular or square cross section, sealed at one end, made of plastic, glass, or fused quartz and designed to hold samples for spectroscopic experiments. The best cuvettes are as clear as possible, without impurities that might affect a spectroscopic reading...
as the reaction platform. The biochip is used to simultaneously analyze a panel of related tests in a single sample, producing a
patientA patient is any person who receives medical attention, care, or treatment. The person is most often ill or injured and in need of treatment by a physician or other health care professional, although one who is visiting a physician for a routine check-up may also be viewed as a patient.The word...
profile. The patient profile can be used in disease screening,
diagnosisIn medicine, diagnosis is a label given for a medical condition or disease identified by its signs, symptoms, and from the results of various diagnostic procedures...
, monitoring disease progression or monitoring treatment. Performing multiple analyses simultaneously, described as multiplexing, allows a significant reduction in processing time and the amount of patient sample required. Biochip Array Technology is a novel application of a familiar methodology, using sandwich, competitive and antibody-capture
immunoassayAn immunoassay is a biochemical test that measures the concentration of a substance in a biological liquid, typically serum or urine, using the reaction of an antibody or antibodies to its antigen. The assay takes advantage of the specific binding of an antibody to its antigen...
s. The difference from conventional immunoassays is that the capture ligands are covalently attached to the surface of the biochip in an ordered array rather than in solution.
In sandwich assays an enzyme-labelled antibody is used; in competitive assays an enzyme-labelled antigen is used. On antibody-antigen binding a chemiluminescence reaction produces light. Detection is by a
charge-coupled deviceA charge-coupled device is a device for the movement of electrical charge, usually from within the device to an area where the charge can be manipulated, for example conversion into a digital value. This is achieved by "shifting" the signals between stages within the device one at a time...
(CCD) camera. The CCD camera is a sensitive and high-resolution sensor able to accurately detect and quantify very low levels of light. The test regions are located using a grid pattern then the chemiluminescence signals are analysed by imaging software to rapidly and simultaneously quantify the individual analytes.
Details about other array technologies can be found in the following pages:
Antibody microarrayAn antibody microarray is a specific form of protein microarrays, a collection of capture antibodies are spotted and fixed on a solid surface, such as glass, plastic and silicon chip for the purpose of detecting antigens...
and
See also
- DNA microarray
A DNA microarray is a multiplex technology used in molecular biology and in medicine. It consists of an arrayed series of thousands of microscopic spots of DNA oligonucleotides, called features, each containing picomoles of a specific DNA sequence...
- Protein array
- Chemical compound microarray
A chemical compound microarray is a collection of organic chemical compounds spotted on a solid surface, such as glass and plastic. This microarray format is very similar to DNA microarray, protein microarray and antibody microarray...
- Antibody microarray
An antibody microarray is a specific form of protein microarrays, a collection of capture antibodies are spotted and fixed on a solid surface, such as glass, plastic and silicon chip for the purpose of detecting antigens...
- Tissue microarray
Tissue microarrays consist of paraffin blocks in which up to 1000 separate tissue cores are assembled in array fashion to allow multiplex histological analysis.-History:...
- Single nucleotide polymorphism
A single-nucleotide polymorphism is a DNA sequence variation occurring when a single nucleotide — A, T, C, or G — in the genome differs between members of a species...
- Sequencing
In genetics and biochemistry, sequencing means to determine the primary structure of an unbranched biopolymer. Sequencing results in a symbolic linear depiction known as a sequence which succinctly summarizes much of the atomic-level structure of the sequenced molecule.-DNA sequencing:DNA...
- Lab-on-a-chip
A lab-on-a-chip is a device that integrates one or several laboratory functions on a single chip of only millimeters to a few square centimeters in size. LOCs deal with the handling of extremely small fluid volumes down to less than pico liters. Lab-on-a-chip devices are a subset of MEMS devices...
- Planar Patch Clamp
- Nanosensors
- Magnetic immunoassay
Magnetic immunoassay is a novel type of diagnostic immunoassay using magnetic beads as labels in lieu of conventional enzymes , radioisotopes or fluorescent moieties . This assay involves the specific binding of an antibody to its antigen, where a magnetic label is conjugated to one element of...