The development of
biochips is a major thrust of the rapidly growing
biotechnologyBiotechnology is a field of applied biology that involves the use of living organisms and bioprocesses in engineering, technology, medicine and other fields requiring bioproducts. Biotechnology also utilizes these products for manufacturing purpose...
industry, which encompasses a very diverse range of
research efforts including
genomicsGenomics is a discipline in genetics concerning 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,...
,
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.
History
The development of bioc 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...
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. It is an important part of the instrumentation for chemical analysis and physico-chemical studies. In modern practice, widely used membranous ion-selective electrodes are part...
, invented in 1922 by
Hughes (Hughes, 1922). Measurement of
pHIn chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
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 , also known as a specific ion electrode , is a transducer that 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...
in subsequent years. For example, a K
+ sensor was
produced by incorporating
valinomycinValinomycin is a dodecadepsipeptide antibiotic.Valinomycin is obtained from the cells of several Streptomyces strains, among which "S. tsusimaensis" and S. fulvissimus....
into a thin membrane (Schultz, 1996).
Over thirty years elapsed before the first true
biosensorA biosensor is an analytical 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 an analytical device for the detection of an analyte that combines a biological...
(
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 is a simple sugar and an important carbohydrate in biology. Cells use it as the primary source of energy and a metabolic intermediate...
sensor developed in 1962 by Clark
and colleague Lyons which utilized
glucose oxidaseThe glucose oxidase enzyme is an oxido-reductase that catalyses the oxidation of glucose to hydrogen peroxide and D-glucono-δ-lactone. In cells, it aids in breaking the sugar down into its metabolites....
molecules embedded in a
dialysisIn medicine, dialysis is a process for removing waste and excess water from the blood, and is primarily used to provide an artificial replacement for lost kidney function in people with renal failure...
membrane (Clark, 1962). The
enzymeEnzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
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 CrickJames D. Watson and Francis Crick were the two co-discoverers of the structure of DNA in 1953. They used x-ray diffraction data collected by Rosalind Franklin and proposed the double helix or spiral staircase structure of the DNA molecule...
announced their discovery of the now familiar
double helix structure of
DNADeoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
molecules and set the stage for
geneticsGenetics , a discipline of biology, is the science of genes, heredity, and variation in living organisms....
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...
techniques in 1977 by
GilbertWalter Gilbert is an American physicist, biochemist, molecular biology pioneer, and Nobel laureate.-Biography:Gilbert was born in Boston, Massachusetts, on March 21, 1932...
(Maxam, 1977) and
SangerFrederick Sanger, OM, CH, CBE, FRS is an English biochemist and a two-time Nobel laureate in chemistry, the only person to have been so. In 1958 he was awarded a Nobel prize in chemistry "for his work on the structure of proteins, especially that of insulin"...
(Sanger, 1977) (working separately) enabled researchers to
directly read the genetic codes that provide instructions for
protein synthesisProtein biosynthesis is the process in which cells build or manufacture 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...
. This research showed how hybridization of complementary single
oligonucleotideAn oligonucleotide is a short nucleic acid polymer, typically with fifty or fewer bases. Although they can be formed by bond cleavage of longer segments, they are now more commonly synthesized, in a sequence-specific manner, from individual nucleoside phosphoramidites...
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 a Nobel Prize winning American biochemist, author, and lecturer. In recognition of his improvement of the polymerase chain reaction technique, he shared the 1993 Nobel Prize in Chemistry with Michael Smith and earned the Japan Prize in the same year. The process was first...
invented the
polymerase chain reactionThe polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a 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...
s instead of
radiolabels (Smith, 1986), thus enabling hybridization experiments to
be observed optically.
The rapid technological advances of the
biochemistryBiochemistry, sometimes called biological chemistry, is the study of chemical processes in living organisms, including, but not limited to, living matter. Biochemistry governs all living organisms and living processes...
and
semiconductorA semiconductor is a material with electrical conductivity due to electron flow intermediate in magnitude between that of a conductor and an insulator. This means a conductivity roughly in the range of 103 to 10−8 siemens per centimeter...
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 that converts one type of energy to another. Energy types include electrical, mechanical, electromagnetic , chemical, acoustic or thermal energy. While the term transducer commonly implies the use of a sensor/detector, any device which converts energy can be considered a...
must be done to translate the actual sensing
event (DNA binding,
oxidation/reductionRedox reactions describe all chemical reactions in which atoms have their oxidation state changed....
,
etc.) into a format
understandable by a computer (
voltageVoltage, otherwise known as electrical potential difference or electric tension is the difference in electric potential between two points — or the difference in electric potential energy per unit charge between two points...
, 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 company that manufactures DNA microarrays; it is based in Santa Clara, California, United States. The company was founded by Dr. Stephen Fodor in 1992. It began 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 tumor suppressor protein that in humans is encoded by the TP53 gene. p53 is crucial 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 caretaker gene that produces a protein called breast cancer type 1 susceptibility protein, responsible for repairing DNA. The first evidence for the existence of the gene was provided by the King laboratory at UC Berkeley in 1990...
and
BRCA2BRCA2 is a protein that in humans is encoded by the BRCA2 gene.BRCA2 orthologs have been identified in most mammals for which complete genome data are available....
(related to breast
cancer) (Cheng, 2001). The chips are produced using microlithography
techniques traditionally used to fabricate integrated circuits (see below).
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 . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
,
RNARibonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
, 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 light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
, 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. The stable balance of attractive and repulsive forces between atoms when they share electrons is known as covalent bonding....
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
CartesianA Cartesian coordinate system specifies each point uniquely in a plane by a pair of numerical coordinates, which are the signed distances from the point to two fixed perpendicular directed lines, measured in the same unit of length...
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 company that manufactures DNA microarrays; it is based in Santa Clara, California, United States. The company was founded by Dr. Stephen Fodor in 1992. It began 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...
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 participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
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 . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
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 , is a popular format of a "wet-lab" type analytic biochemistry assay that uses one sub-type of heterogeneous, solid-phase enzyme immunoassay to detect the presence of a substance in a liquid sample."Wet lab" analytic biochemistry assays involves detection of an...
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 recipient of healthcare services. The patient is most often ill or injured and in need of treatment by a physician, advanced practice registered nurse, veterinarian, or other health care provider....
profile. The patient profile can be used in disease screening,
diagnosisMedical diagnosis refers both to the process of attempting to determine or identify a possible disease or disorder , and to the opinion reached by this process...
, 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 presence or concentration of a substance in solutions that frequently contain a complex mixture of substances. Analytes in biological liquids such as serum or urine are frequently assayed using immunoassay methods...
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 page:
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...
See also
- DNA microarray
A DNA microarray is a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome...
- 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 biological species or paired chromosomes in an individual...
- Sequencing
In genetics and biochemistry, sequencing means to determine the primary structure of an unbranched biopolymer...
- 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...