China Medical University Student ePaper2

Microarray and bio-chips
1 China Medical University Student ePaper
China Medical University Student ePaper
Title：Microarray and bio-chips
By Yi Han Chiu
Required Course: Biotechnology seminar in Department of
Medical Laboratory Science and Biotechnology
Approved: 2 semester credits
Advisor: Meng-Liang Lin, Associate Professor

Abstract
(1) Objective: Microarray biochip is the products that using the method of
semiconductor, such as pin and injects printing. It is the brand new laboratory
evolution to analyze the sample in an instant, automatic and high efficiency way.
In recently, microarray biochip has a prosperous future in many areas, for example,
doctors can make a quick detection in disease control and prevention. Moreover, in
pharmaceutical company, technicians can develop the new drugs with microarray
biochip to having a fast and specific quality.
(2) Method: In microarray biochips, it can be divided into two parts, DNA chips and
protein chips. DNA chips used nucleic acid as probe, mainly for the analysis of gene
sequences. Each probe is different from others, therefore, DNA chips can examined
twenty thousand more different genes in the same time. On the other hand, protein
chips is the new development after DNA chips, normally used proteins, antibodies or
microorganisms as probe, processing antibody-antigen immune response,
importantly detect hormones, clotting factors and biological functional performance.
(3) Result:
Microarray biochip is a latest technology that can put many sample, reagent and
biological materials in a tiny, miniaturized device. These biological materials react
specific biochemical with other substances, after quantitatively analysis, the data
may appear immediately. Only needs a few sample, microarray biochip can detect a
variety of different diseases. Thus, Microarray biochip become developed target
technology in medical diagnostics, drug development.
Keyword：
Biochip
Single nucleotide polymorphism (SNP)
Medical technology
Microarray
Microfluidic

Contents
 Introduction
 History
 Types of Biochips
 Principle of Microarray Chips
 Microarray Technologies
 Detection
 Application of Microarray Chips
 Development in Taiwan
 Reference
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Introduction
Transcription
Collect samples→
Amplification
Microarray chips → Ligand attachment wash
Photolithography
Pin printing
Dip-pen nanolithography detection
Ink-jet printing
Light-directed synthesis data analysis
Genome mapping provided a critical starting point for the Human Genome Project.
With the completion of the human genome sequence and analysis gene structure,
scientists find out the connection between each human genes and protein function.
Combining microelectronics, micro-electromechanical systems, life sciences and
bio-information with biochip industry may accelerate the speed of decoding. Bio-chip
covers a wild range of functional genomics research including new drug development,
clinical testing, screening, environmental controls, and so on. This means that 21st
century will be the century of bio-chip development and increasingly become a
new mainstream.
Biological targets
extraction
Probe-target
hybridization

History
Micro array development has become a large number of screening technologies,
mainly extend from two types of molecular biology and view the earliest microarray.
The first of these technologies enzyme-linked immunosorbent assay; ELISA which can
simultaneously to 96 different sample detect specific protein; the second technology
is dot blotting assay mainly detect specific nucleic acid sequence. As biometric
technology breakthroughs, precision measure tools development and automated
facilities can be scaled down more accurate and keep it in small range detection for
tens of thousands of gene become available.
In 1980, Caras and Janata demonstrated the earliest biochip that an immobilized
penicilinase layer over the gate insulator of the ISFET (Ion-Sensitive Field Effect
Transistor) could be used as a penicillin sensor. While the penicillin acid attach to the
acid and alkaline sensor membrane, it generates a power-change which evaluated
the volume of penicillin by the output signal.
In 1991, Affymax Research developed a new technology that uses solid surface
chemistry, photo labile protecting groups and photolithography. The initial
microarray consisted of 1024 peptides in a 1.6 cm2
area and can be used to detect
single monoclonal antibodies qualification. 3 Years later by the same research team
with the same way used photolithography synthesis the high-density nucleic acid
directly into the chip, called the DNA chip.
1995 former Stanford University Professors believe that the concept of high-density
nucleic acid arrange neatly on the chips can be used to analyze DNA performance.
Next year, Affymetrix Company merchandise the DNA chips and launches to add this
item named Gene chip. This microarray technology is no longer confined the probe
to cDNA or oligonucleotide, but includes all life sciences on important molecules or
substances can serve as probe materials.

Types of Biochips
• Microarray
Microarray chip include: DNA chip and protein chip.
1. DNA chip：
A. Oligonucleotides microarray (ONA)
B. cDNA microarray (CDA)
A. Oligonucleotides microarray (ONA)：The in situ synthesis of oligonucleotides
occur in parallel, resulting in consecutive addition of A, C, G and T
nucleotides to the appropriate gene sequences on the array. Oligonucleotide
chains that for example require adenine in the next position are protected
by light at the appropriate positions by a solid support mask which made up
of nylon filter or glass slide. Physical masking using mechanical flow cells
and conventional synthetic chemistry gives high coupling yields and
provides high resolution, better used for sets of probes with related
sequences, a gene of known sequence. Moreover, ink jet fabrication is rapid,
highly flexible and has a high throughput.
B. cDNA microarray (CDA)：cDNA microarray is a set of short Expressed
Sequence Tags (ESTs) made from a cDNA library of a set of known (or
partially known) gene loci. The ESTs are spotted onto a cover-slip-sized glass
plate, shown as a 8x12 array and the nucleic acid length range 500-5000
mer.
Bio-chip
Microarray
DNA chip Protein chip
Microfluidic
chip
Lab-on-a
chip

Oligonucleotides microarray(ONA) cDNA microarray(CDA)
Chip density High density Medium density
Length <25 mer 500～5000 mer
Application oligonucleotides microarray DNA、antigen-antibody、drug
receptor
Features Useful for genome analysis,
map-based cloning, promoter
studies
Useful for analysis of coding
regions and gene functions
sequencing、point mutation、
sequences interaction
Gene express、gene mapping
Source : spot your genes an overview of the microarray(2004). Retrieved from:
http://www.scq.ubc.ca/spot-your-genes-an-overview-of-the-microarray/
2. Protein chip
Protein microarrays, also known as protein chips, are miniaturized and parallel
assay systems that contain small amounts of purified proteins in a high-density
format within a single experiment. Protein microarrays are typically prepared by
immobilizing proteins onto a microscope slide using a standard contact spotter or
noncontact microarray. Analytical protein arrays can be used to monitor protein
expression levels or for biomarker identification, clinical diagnosis, or
environmental safety analysis. Functional protein microarrays have many uses:

including protein-protein, protein-lipid, protein-DNA, protein-drug, and
protein-peptide interactions; to identify enzyme substrates; and to profile
immune responses, among many others.
Source: Zhong et al. BMC Biotechnology 2010. Retrieved from
http://php.med.unsw.edu.au/cellbiology/index.php?title=File:Z3269335_ProteinMicroarrayVs_ELISA.j
pg
3. Lab on a chip-LOC
The biochemistry detection usually use traditional methods, for example: after
venous puncture, the blood is divided into different tube and mix, centrifugal, culture,
heat, duplicate, etc. These steps in the general chemistry lab is divided into several
different instruments and require very large space, time-consuming and requires a
large amount of the specimen. However, Lab on a Chip provides a unique form for
the publication of significant and original work related to miniaturization (on or off
chips) at the micro- and nano-scale across a variety of disciplines. Miniaturization of
biochemical operations normally handled in a laboratory has numerous advantages
such as cost efficiency, parallelization, diagnostic speed and sensitivity. The
emergence of the lab-on-a-chip field mainly relies on two core technologies:
microfluidics and molecular biology.

Source : Burns, M.A. et al. “An Integrated Nanoliter DNA Analysis Device,” Science 282: 484-487, 1998.
Retrieved from http://lsi.epfl.ch/page-13122-en.html
4. Microfluidic chip
Microfluidic chip also called CE-Chip. It is a set of micro-channels molded into a
material (glass, silicon or polymer such as PDMS, for Poly Dimethyl Siloxane). The
micro-channels forming the microfluidic chip are connected together in order to
achieve the desired features (mix, pump, sort, control bio-chemical environment).
This network of micro-channels trapped into the microfluidic chip is connected to
the outside by inputs and outputs pierced through the chip, as an interface between
the macro- and micro-world. It is through these holes that the liquids (or gases) are
injected and removed from the microfluidic chip (through tubing, syringe adapters or
even simple holes in the chip) with external active systems. In this system use
electro-osmosis, capillary and pressure balance with voltage or air pressure to
analysis electrophoresis。
Caliper Technologies Corp., for instance, Caliper's mobility shift assays can be used to
investigate a wide range of drug targets. Mobility shift assays rely on the separation
of reactants from products and reading the fluorescence of each to determine the
extent of the reaction. As a result, there is no need for complex biochemical
reactions, specialized antibodies, complicated labeling strategies or the use of
radioactivity. Unlike these other approaches, only a fluorescently labeled peptide
substrate is needed to perform the reaction and separation in the Caliper format.
This provides more accurate results and reduced assay-development time.

Source : retrieved from http://www.nanosyn.com/?id=40&page=biology-technology

Principle of Microarray Chips
The silicon chip has a gold-based surface layer (gold’s biological compatibility better)
and a cloth layer connection-level (Organic materials), easily for each strand of DNA
attach on.
Probe DNA: DNA with known bases A, T, C, G serial fixed on the silicon chip.
Target DNA: Also called detected DNA, Contains Unknown A, T, C, G serial, in order to
know that target DNA bind to probe DNA with hydrogen bond, usually add
fluorescent substances in the end of target DNA and observe under the microscope.
The semiconductor manufacturing process such as photo-mask, lithography, etched
methods. The probe DNA of A, T, C, G fix on the silicon chip and each point on the
array is only 1 µm. Each probe DNA has different A, T, C, G sequence. When DNA
hybridization starts, suppose there is three points emit fluorescence, which sequence
are GCAC, GTTG, TGGG. As the result, the target DNA sequence are CGTG, CAAC,
ACCC.
Source: Making Chips to Probe Genes(2001)
retrieved from http://spectrum.ieee.org/biomedical/diagnostics/making-chips-to-probe-genes.

Microarray Technologies
Microarrays can be fabricated by a variety of technologies, including printing with
fine-pointed pins onto glass slides, photolithography using pre-made masks, ink-jet
printing, and electrochemistry on microelectrode arrays.
A. Pin printing：
The pin tools are dipped into DNA sample, and transfer a small amount of
sample onto the tip of the pins. Then, leave a spot on the chip surface when
touching the pin. It is relatively easy to pin 10000 cDNAs onto the chip
(3.6cm2
). The pin technology is fast, affordable and provides multi-function.
Disadvantage is that each sample must be composite and purify. 。
B. inject printing：
Inject printing technology uses a piezoelectric crystal closely opposed to the
fluid reservoir. The reservoir and the crystal are based on a voltage, which
causes the crystal to deform, squeeze the capillary and eject a small amount
of fluid from the tip. Sample could be cDNAs, chromosome DNAs or antibody.
The difference between pin and inject printing is the orifice do not attach the
chip. Although it is relatively fast, the price is higher due to micro-and
multi-samples.
C. in situ synthesized：
Photolithography and DNA hybridization are the methods to fix four types of
DNA pattern which is link with different pendant groups. Though the
substance with photoresist on the bottom, covered with a mask, and exposed
to ultraviolet (UV) irradiation. DNA synthesis could be done on the chip
(1.6cm2
) by a small amount of nucleic acid (400kp) and reagent. This
technology might be better than the two methods above either synthesis
density or precision.

Source: Making Chips to Probe Genes(2001)
retrieved from http://spectrum.ieee.org/biomedical/diagnostics/making-chips-to-probe-genes

Detection
Device to analysis the results of the chip should according to the types of molecules.
The earliest detection is isotopic labeling, requiring exposure, development, and then
use a scanner provides location-finding system.Fluorescent label is the most common
and most successful in collecting data. This method does not have isotope restriction.
Using laser excitation fluorescent in display hybridization results that have a very high
level of differentiation can quantify the results; include a high sensitivity and
positioning features. First, insert the chip in the scanner, after reading approximately
3 minutes/tab, the software calculate the points of light position (DNA site),
fluorescent strength, fluorescent contrast and scale, distribution curve. For example,
protein chip can use protein as a probe that fixed on the surface of the substance.
Then adding unknown sample and conduct antigen-antibody immunoreaction with
secondary antibody combine fluorescent substance. After washing out the unbinding
secondary antibody, the chip may insert in the scanner to detect the specificity of the
sample. Recently, other technology such as SELDI（Surface Enhanced Laser
Desorption/Ionization）and SPR（Surface Plasmon Resonance） are increasingly
common in use.
Source : retrieved from Microfabrication of patterns of adherent marine bacteria Phaeobacter
inhibens using soft lithography and scanning probe lithography. Langmuir 2010, 26, 8641-8647.
SELDI（Surface Enhanced Laser
Desorption/Ionization）
SPR（Surface Plasmon Resonance）
Description proteins of interest in a sample
become bound to a surface before
MS analysis. SELDI is typically used
with time-of-flight (TOF) mass
spectrometers and is used to detect
proteins in tissue samples.
The Surface Plasmon Resonance (SPR)
is an Optical technology for
monitoring specific binding events
between two or more bio-molecules
without the use of labels.
Features Analysis protein molecular weight Provide Real-time Monitoring of

Biomolecule Interaction and Protein
and DNA Linker Characterization.
Application Diagnose and toxicology screening Research kinetics of protein reactions
Application of Microarray Chips
The current medical technology only until the confirmation of disease has been made
toward the diagnosis and treatment. Clinical has many advanced diagnostic
instruments, but these instruments resolution can only be reached 0.5~1.0cm, for
the molecular level of variable cannot be effectively solve, particularly molecules
image information. However, due to biochip, not only observe hundreds of even
thousands of DNA or protein performance with a medical imaging, but also provide
cells of abnormal signs and Disease symptoms. Biochip technology can quickly find
the target gene to develop genetic probe to create molecular images, which would
benefit the disease treatment and the pre-diagnosis stage. Biochip can be the most
important tools in medical research.
Biochip applications are widely used, for example:
1. Gene detection:
Through analysis the DNA in different organizations, different development stage of
performance and determine its features.
2. Cancer research:
Microarray can identify the mutation of proto-oncogene or tumor suppression.
3. New Drug Development:
DNA microarray can analyze the genetic changes of treatment so it can be used to
evaluate the cells reaction during the new drugs.
4. Single nucleotide polymorphism (SNP):
SNP is a variation in a single nucleotide that occurs at a specific position in the
genome, if more than 1% of a population does not carry the same nucleotide at a
specific position in the DNA sequence, and then this variation can be classified as a
SNP. SNP also lead to the sensitivity in cancer, new vessel disease, self-immunity and
diabetes. By designing a set of that contains a different sequence of probe DNA,
human SNP could possibly be identified.

5. Clinical Exam: Health care, pathogen detection, blood screening, and so on.
6. Non-medical applications: such as military detection, personal identification,
criminal scene investigation, environment and food exams, and so on.
Source: Analysis of Global Biochips Industry(2014)
Retrieved from
http://www.giichinese.com.cn/report/arm275350-analysis-global-biochips-industry.html

Development in Taiwan
Recent advancements in several genome-sequencing projects have stimulated an
enormous interest in microarray DNA chip technology, especially in biomedical
sciences and pharmaceutical industries in Taiwan. The current status of biochip
projects in Taiwan, such as protein chip, PCR chip, electrophoresis chip, olfactory
chip, etc. The new development of biochip technologies integrates the
biotechnology with the semiconductor processing, the micro-electro-mechanical,
optoelectronic, and digital signal processing technologies. Most of these biochip
technologies utilize optical detection methods for data acquisition and analysis.
1. Phalanx Biotech Group:
Founded in 2002, Phalanx Biotech Group was created by several leaders from the
highly regarded Industrial Technology Research Institute (ITRI) of Taiwan. In 1998, the
Biomedical Engineering Center (BMEC) within ITRI initiated the Biochip Program to
explore the potential of microarray technology. Because of the success of this
program, the team’s combined technical expertise produced key advancements in
micro fluids and microarray manufacturing. The team generated over 15 patents,
which are licensed exclusively by Phalanx Biotech Group in order to promote growth
in the microarray technology industry.
Source:Phalanx Biotech Group
Retrieved from：http://www.phalanx.com.tw/OEM/OEM_Power.php
2. Excelsior Bio-System, Inc:
Excelsior Bio-System (EBS) was founded in October 2004 by Dr. Martin Hsiao and Mr.
K.J. Chen with the goal of developing alternative therapy products, holistic natural
food supplements and probiotics, and creating a series of diagnostic systems using
biochip detection technology to detect immune system related diseases in both
people and animals. From 2004 to 2007, the company utilized some of these core

technologies—including allergen protein extraction, fluorescence-labeling of
antibodies, protein chip platforms, and functional probiotics—to develop a fully
functional allergen-specific IgE diagnostic chip, self-antigen IgG autoimmune disease
test chip prototype, as well as various immune-regulating probiotics, and then
subsequently carried out functional testing on these newly-developed prototypes.
 Excelsior Bio-System, Inc.
Retrieved from：http://www.ebs.com.tw/en/company/about
Reference
 Li-Qiang Ma, Zhong-Hua Jiang (2003)。Biochip。Taipei : Jeou Chou Book Co, Ltd.
(ISBN 9578324782)
 Dr. Kai-An Cheng, Nai-Ching Yeh, Jeng-Hua Yin, Hai-Chen Lin (2003). The
Opportunity of biochip industry in Taiwan. Hsinchu: Industrial & Technology
Intelligence Services (ITIS). (ISBN 9577745415)
 Lai, Chee-yuen(2005). Technology Roadmapping of Microfluidic Biochip.
Taipei:Science & Technology Policy Research and Information Center, STPI.
(ISBN 9576191181)
 William J. Thieman, Michael A. Palladino(2004).Introduction to
Biotechnology.San Francisco : Pearson/ Benjamin Cummings, c2004. (ISBN
0321766113)
 Chien-Min Kung (2009).Introduction of biotechnology. Taichung: Wagners
publishing Co., Ltd. (ISBN 9789863622031)
 Sharon Walker (2006). Biotechnology Demystified. London, United
Kingdom:McGraw-Hill Education. (ISBN 0071448128)
 Nam-Trung Nguyen, Steven T. Wereley, “Fundamentals and applications of
microfluidics”, Boston, MA :Artech House,c2002 (ISBN 1580533434)
 Patrick Tabeling; translated by Suelin Chen, “Introduction to microfluidics”,
Oxford, U.K.; Oxford University Press, 2005, New York (ISBN 0198568649)

 Phalanx Biotech Group
Retrieved from：http://www.phalanx.com.tw/OEM/OEM_Power.php
 Excelsior Bio-System, Inc.
Retrieved from：http://www.ebs.com.tw/en/company/about
 The new tools to enhance drug development efficiency－the protein chip
development (2001).
Retrieved from：
http://www.moneydj.com/kmdj/report/ReportViewer.aspx?a=df1be3cd-80d8
-45e2-9b2f-4f1764fe0666#ixzz2xSeb0QOE

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