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New Electrostatic-based DNA Microarray Technique

The dream of personalized medicine — in which diagnostics, risk predictions and treatment decisions are based on a patient's genetic profile — may be on the verge of being expanded beyond the wealthiest of nations with state-of-the-art clinics. A team of researchers with the U.S. Department of Energy's Lawrence Berkeley National Laboratory (Berkeley Lab) has invented a technique in which DNA or RNA assays — the key to genetic profiling and disease detection — can be read and evaluated without the need of elaborate chemical labeling or sophisticated instrumentation. Based on electrostatic repulsion — in which objects with the same electrical charge repel one another — the technique is relatively simple and inexpensive to implement, and can be carried out in a matter of minutes.

"One of the most amazing things about our electrostatic detection method is that it requires nothing more than the naked eye to read out results that currently require chemical labeling and confocal laser scanners," said Jay Groves, a chemist with joint appointments at Berkeley Lab's Physical Biosciences Division and the Chemistry Department of the University of California (UC) at Berkeley, who led this research. "We believe this technique could revolutionize the use of DNA microarrays for both research and diagnostics."

Groves, who is also a Howard Hughes Medical Institute (HHMI) investigator, and members of his research group Nathan Clack and Khalid Salaita, have published a paper on their technique in the journal Nature Biotechnology, which is now available online. The paper is entitled "Electrostatic readout of DNA microarrays with charged microspheres."

In their paper, Groves, Clack, and Salaita describe how dispersing a fluid containing thousands of electrically-charged microscopic beads or spheres made of silica (glass) across the surface of a DNA microarray and then observing the Brownian motion of the spheres provides measurements of the electrical charges of the DNA molecules. These measurements can in turn be used to interrogate millions of DNA sequences at a time. What's more, these measurements can be observed and recorded with a simple hand-held imaging device — even a cell phone camera will do.

"The assumption has been that no detection technique could be more sensitive than fluorescent labeling, but this is completely untrue, as our results have plainly demonstrated," said Groves. "We've shown that changes in surface charge density as a result of specific DNA hybridization can be detected and quantified with 50-picomolar sensitivity, single base-pair mismatch selectivity, and in the presence of complex backgrounds. Furthermore, our electrostatic detection technique should render DNA and RNA microarrays sufficiently cost effective for broad world-health applications, as well as research."

Your susceptibility to a given disease and how your body will respond to drugs or other interventions is unique to your genetic makeup. Under a personalized medicine plan, treatment effectiveness is maximized and risks are minimized by tailoring disease treatments specifically to you. This requires the precise diagnostic tests and targeted therapies that can stem from assays using a DNA microarray — a thumbnail-sized substrate containing thousands of microscopic spots of oligonucleotides (stretches of DNA about 20 base pairs in length) laid out in a grid.

Often referred to as "gene chips," DNA microarray assays and their RNA counterparts have become one of the most powerful tools for gene-expression profiling, the identification of mutations, and the detection of multiple pathogens in patients afflicted either by multiple diseases or drug-resistant strains of diseases. Aside from their potential future role in personalized medicine, the widespread use of DNA microarray assay devices could have an immediate and profound impact on the treatment of diseases today. For example, according to a report two years ago from the Global Health Diagnostics Forum, 400,000 lives could be saved each year from death by tuberculosis through the use of DNA microarray assays rather than the standard TB diagnostic test.

We have demonstrated parallel sampling of a microarray surface with micron-scale resolutions over centimeter-scale lengths," said Groves. "This is four orders of magnitude larger than what has been achieved to date with conventional scanning-electrostatic-force microscopy."

In a typical experiment, a microarray is prepared and mounted in a well chamber and the DNA is hybridized (a standard technique in which complementary single strands of DNA bind to form double-stranded DNA "hybrids"). A suspension of negatively-charged silica microspheres is then dispersed through gravitational sedimentation over the microarray surface, a process which takes about 20 minutes. Because the substrate or background surface of the microassay is positively charged, the silica microspheres will spread across the entire surface and adhere to it. However, on surface areas containing double-stranded DNA, which is highly negatively charged, and on areas containing single-stranded DNA, also negatively charged but to a lesser degree than double-stranded DNA, the microspheres will levitate above the substrate surface, stacking up in "equilibrium heights" that are dictated by a balance between gravitational and electrostatic forces.

These electrostatic interactions on the microarray surface result in charge-density contrasts that are readily observed. Surface areas containing DNA segments take on a frosted or translucent appearance, and can be correlated to specific hybridizations that reveal the presence of genes, mutations and pathogens.

There are a number of short-term "next steps" for this research, Groves said, including testing its application in high-density arrays and pushing its ultimate resolution limits.

"Since the resolution of electrostatic-based imaging is determined by the number of particle-observations rather than by the diffraction limit of light, our readouts could serve as a form of ultramicroscopy," he said. "The real grand challenge for this technology, however, will be for us to find suitable industrial partners with whom we can work to see that useful new products actually make it to market.

DNA Microarray picture

A turning point in Science and our approach to the human body occurred in 1953 when Watson and Crick discovered the double helix structure of DNA. DNA consists of four base chemicals called nucleotides: adenine, guanine, cytosine and thymine that reside on the inner surface of two parallel sugar and phosphate rails. DNA holds all the information required to produce and maintain a living organism.

In 2003 the Human Genome Project was completed and the entire sequence of human genes was unraveled, mapped and stored in databases. In the past decade DNA microarray technology has made it possible to measure the expression levels of many thousands of genes simultaneously.

DNA microarrays are quite complex, but basically thousands of microscopic spots of DNA can be attached to a solid surface, usually glass or silicon. This gives rise to the name “gene chip”. A gene chip can be analyzed for coding structure. The coding structure can then be referenced against the knowledge that exists on the gene databases. The genes can also be referenced to a person’s own DNA to observe changes and differences over time.

A recent article in the Australian Journal of Clinical Hypnotherapy & Hypnosis made me aware of the existence of a branch of research entitled Psychosocial Genomics. Psychosocial Genomics is the study of how inner psychological events and interactive social events can stimulate gene expression. The process is thought to proceed from brain to body.

Until I read this article I had no idea that such research has ever taken place and I was immediately fascinated by it. I had been practicing Clinical Hypnotherapy since 1993, knowing that it achieved amazing results but not thoroughly understanding how these results were achieved. I had too much scientific training behind me to be caught up in the egoistic notion that it was just I who was responsible for the results. I knew instinctively that changes must take place in the clients’ neurotransmitters levels, but further than that, did not give it further thought.

Reading the article revealed to me that Ernest Rossi, a leading Psychotherapist and Clinical Psychologist had proposed a connection between gene expression, protein synthesis and hypnosis as far back as the mid-seventies.

Psychosocial Genomics, through the examination of gene expression seeks to understand the activity of the hypnotherapeutic process and provide some degree of measure. Gene expression enables an ongoing cascade of processes in the body including protein synthesis, and a swathe of biological processes. The biological processes include positive healing activities of the immune system (anti-inflammatory), beneficial alteration of mental and emotional states (hormonal, peptide and neurotransmitter balance) and the growth and development of new, neural and neuronal structures (nerve and brain plasticity.

DNA Microarrays Methods Express, Mark Schena, Ph.D.

DNA Microarrays – Methods Express, edited by Mark Schena and published by Scion Publishing. Methods book containing 12 chapters from leading laboratories covering the latest protocols and procedures for DNA microarray experimentation. This must-have book is an essential resource for all laboratories working on DNA microarrays.

Electronic DNA microarrays

DNA contains and issues the language of life. It gives cells instructions for living, and tells living organisms about their hereditary traits. This language is coded into the DNA’s famous double helix structure: Fig 1(a). Each helical strand exhibits a sequence of four chemical bases, adenine (A), guanine (G), cytosine (C), & thymine (T), e.g., CAAGTG. The two twisted strands are bound together by pairing base A always with base T, and G always with C. Due to this pairing rule, within any section of DNA, once the sequence of one strand is identified, that of the other strand is easily inferred: e.g., the complementary sequence of CAAGTG is GTTCAC. DNA sequences are the language of life. Reading them, therefore, is of prime importance.
    Electronic DNA microarrays are CMOS integrated circuits (ICs) that can rapidly decipher unknown DNA sequences [1-6]. A double-stranded DNA molecule can unzip into two complementary strands. A single-stranded DNA molecule thus obtained can bind back to its complementary sequence (either the old mate or a new one), forming again a double-stranded DNA molecule. This binding of two complementary strands, or hybridization, underlies the genetic sequencing operation of the electronic DNA chip.

The electronic DNA microarray is constructed by immobilizing single-stranded DNA molecules of different identified sequences onto different grid points on a CMOS IC [Fig. 1(b)]. The grid points are often defined by post-fabricated gold electrodes that are electrically connected to the underlying CMOS IC. Different grid points represent distinct DNA sequences. These single-stranded DNA molecules of known sequences making up the array are called DNA probes. Now consider single-stranded DNA molecules of an unknown sequence, or DNA targets. When a solution of DNA targets is introduced onto the DNA array, the target strands wander around to eventually hybridize to their complementary probe strands at a specific grid point [Fig. 1(b)]. Locating the hybridization position reveals the target sequence, for we already know the probe sequence at that position, which must be complementary to the target sequence. The CMOS IC underneath is used for electronic detection of the hybridization point.
One well-established electronic detection technique is to label DNA targets with reporter molecules of a distinctive electronic signature and to search for them. Redox enzymes are an example of such electronic labels. If a voltage is suddenly applied between an electrode where hybridization occurred and the electrolyte (DNA target solution), redox labels attached to target molecules will give up electrons to the electrode, thereby increasing the current through the electrode. The same voltage step in an electrode with no hybridization would cause no such current increase as redox labels are absent at that electrode. By applying the voltage step and monitoring current change fast across the whole array using the underlying IC, hybridization positions are rapidly detected, leading to target sequence identification. Redox-label-based CMOS DNA chip examples are found in [1-3].
While the label-based detection offers excellent sensitivity, significant efforts are being placed to develop label-free electronic DNA microarrays [4,5] because elimination of labeling steps would simplify the sample preparation. In [4], for example, the capacitance of an electrode immersed in the electrolyte is monitored to sense hybridization. No labeling is needed, as target strands added to an electrode during hybridization naturally lead to a dielectric constant change, or, capacitance change.
Field effect transistors (FETs), the basic commodity of CMOS ICs, can be also useddirectly for label-free electronic detection of DNA hybridization [7-9]. Underlying this sensing modality is the exploitation of the impact of DNA’s intrinsic negative charges upon the FET behavior. Imagine that underneath the DNA array there is a corresponding FET array integrated in the CMOS IC. The gate dielectric of each FET is linked to DNA probe strands of the same sequence in a corresponding site in the DNA array. When a target strand hybridizes to its complementary probe strand anchored to a specific FET gate, the target’s intrinsic charge alters the channel conductance and capacitance of the FET. Therefore, by monitoring the channel of each FET in the array, one can attain label-free electronic readout of target sequences. While field effect sensorsare widely used [9], there is a lot of room for development in their use in DNA microarrays. Post-processing to expose gate dielectrics to electrolyte may pose a challenge. The smallest possible FET width must be used to maximize the impact of the DNA’s charge on channel properties.
Hybridization is at the heart of many other DNA sequencing techniques. What makes electronic DNA microarrays unique is their massively parallel operation. Distinct probe sequences numbering as many as hundreds of thousands can be simultaneously available across an array. The CMOS IC monitors each site of the array fast across at a gigahertz speed, and hence, its operation may be regarded as parallel to human eyes. This parallelism allows for rapid collection of vast amounts of genetic information (far faster than non-microarray techniques), accelerating the speed at which we probe the secrets of living organisms. The parallelism is a direct outcome of using CMOS microfabrication techniques to build large microarrays, and is enhanced by the use of integrated electronics.
In the original invention, which is still the commercially dominant form of the DNA microarray, hybridization is sensed by optical means [10]: florescent dyes labeling target strands light up upon illumination, reporting hybridization points. This optical machine boasts sensitivity superior to, and parallelism similar to, its much smaller electronic cousin. Although considerable work is needed to develop a high-performance electronic DNA chip as an alternative to the optical type, decisive advantages of using ICs (small size, low cost, programmability, real-time, label-free options) are the cogent reason for the ever-growing efforts in the development of electronic DNA chips.


Other electronic biosensor microarrays

Generalizing the concepts of the electronic DNA chip, one can readily consider CMOS biosensors that can detect other biological objects such as viruses and disease marker proteins [11]. Just like a DNA strand sticks specifically to its complementary strand, a virus or a protein binds specifically to its unique biochemical mate, an antibody. This highly specific binding is analogous to the way different keys fit into different locks. Fig. 2 illustrates a scenario where nine different types of viruses can be detected by using an array of nine specifically corresponding types of antibodies. When the array is exposed to a target solution containing type-4 viruses, for instance, the viruses will specifically bind to antibodies in array point 4. This binding can be detected using the CMOS IC below in much the same ways DNA hybridization is detected. By reading the location of the binding, the presence and type of viruses are determined. The multiplexed array platform can be especially useful for the diagnostics of complex diseases like cancer.

Electronic DNA microarrays

DNA contains and issues the language of life. It gives cells instructions for living, and tells living organisms about their hereditary traits. This language is coded into the DNA’s famous double helix structure: Fig 1(a). Each helical strand exhibits a sequence of four chemical bases, adenine (A), guanine (G), cytosine (C), & thymine (T), e.g., CAAGTG. The two twisted strands are bound together by pairing base A always with base T, and G always with C. Due to this pairing rule, within any section of DNA, once the sequence of one strand is identified, that of the other strand is easily inferred: e.g., the complementary sequence of CAAGTG is GTTCAC. DNA sequences are the language of life. Reading them, therefore, is of prime importance.
    Electronic DNA microarrays are CMOS integrated circuits (ICs) that can rapidly decipher unknown DNA sequences [1-6]. A double-stranded DNA molecule can unzip into two complementary strands. A single-stranded DNA molecule thus obtained can bind back to its complementary sequence (either the old mate or a new one), forming again a double-stranded DNA molecule. This binding of two complementary strands, or hybridization, underlies the genetic sequencing operation of the electronic DNA chip.

The electronic DNA microarray is constructed by immobilizing single-stranded DNA molecules of different identified sequences onto different grid points on a CMOS IC [Fig. 1(b)]. The grid points are often defined by post-fabricated gold electrodes that are electrically connected to the underlying CMOS IC. Different grid points represent distinct DNA sequences. These single-stranded DNA molecules of known sequences making up the array are called DNA probes. Now consider single-stranded DNA molecules of an unknown sequence, or DNA targets. When a solution of DNA targets is introduced onto the DNA array, the target strands wander around to eventually hybridize to their complementary probe strands at a specific grid point [Fig. 1(b)]. Locating the hybridization position reveals the target sequence, for we already know the probe sequence at that position, which must be complementary to the target sequence. The CMOS IC underneath is used for electronic detection of the hybridization point.
One well-established electronic detection technique is to label DNA targets with reporter molecules of a distinctive electronic signature and to search for them. Redox enzymes are an example of such electronic labels. If a voltage is suddenly applied between an electrode where hybridization occurred and the electrolyte (DNA target solution), redox labels attached to target molecules will give up electrons to the electrode, thereby increasing the current through the electrode. The same voltage step in an electrode with no hybridization would cause no such current increase as redox labels are absent at that electrode. By applying the voltage step and monitoring current change fast across the whole array using the underlying IC, hybridization positions are rapidly detected, leading to target sequence identification. Redox-label-based CMOS DNA chip examples are found in [1-3].
While the label-based detection offers excellent sensitivity, significant efforts are being placed to develop label-free electronic DNA microarrays [4,5] because elimination of labeling steps would simplify the sample preparation. In [4], for example, the capacitance of an electrode immersed in the electrolyte is monitored to sense hybridization. No labeling is needed, as target strands added to an electrode during hybridization naturally lead to a dielectric constant change, or, capacitance change.
Field effect transistors (FETs), the basic commodity of CMOS ICs, can be also useddirectly for label-free electronic detection of DNA hybridization [7-9]. Underlying this sensing modality is the exploitation of the impact of DNA’s intrinsic negative charges upon the FET behavior. Imagine that underneath the DNA array there is a corresponding FET array integrated in the CMOS IC. The gate dielectric of each FET is linked to DNA probe strands of the same sequence in a corresponding site in the DNA array. When a target strand hybridizes to its complementary probe strand anchored to a specific FET gate, the target’s intrinsic charge alters the channel conductance and capacitance of the FET. Therefore, by monitoring the channel of each FET in the array, one can attain label-free electronic readout of target sequences. While field effect sensorsare widely used [9], there is a lot of room for development in their use in DNA microarrays. Post-processing to expose gate dielectrics to electrolyte may pose a challenge. The smallest possible FET width must be used to maximize the impact of the DNA’s charge on channel properties.
Hybridization is at the heart of many other DNA sequencing techniques. What makes electronic DNA microarrays unique is their massively parallel operation. Distinct probe sequences numbering as many as hundreds of thousands can be simultaneously available across an array. The CMOS IC monitors each site of the array fast across at a gigahertz speed, and hence, its operation may be regarded as parallel to human eyes. This parallelism allows for rapid collection of vast amounts of genetic information (far faster than non-microarray techniques), accelerating the speed at which we probe the secrets of living organisms. The parallelism is a direct outcome of using CMOS microfabrication techniques to build large microarrays, and is enhanced by the use of integrated electronics.
In the original invention, which is still the commercially dominant form of the DNA microarray, hybridization is sensed by optical means [10]: florescent dyes labeling target strands light up upon illumination, reporting hybridization points. This optical machine boasts sensitivity superior to, and parallelism similar to, its much smaller electronic cousin. Although considerable work is needed to develop a high-performance electronic DNA chip as an alternative to the optical type, decisive advantages of using ICs (small size, low cost, programmability, real-time, label-free options) are the cogent reason for the ever-growing efforts in the development of electronic DNA chips.


Other electronic biosensor microarrays

Generalizing the concepts of the electronic DNA chip, one can readily consider CMOS biosensors that can detect other biological objects such as viruses and disease marker proteins [11]. Just like a DNA strand sticks specifically to its complementary strand, a virus or a protein binds specifically to its unique biochemical mate, an antibody. This highly specific binding is analogous to the way different keys fit into different locks. Fig. 2 illustrates a scenario where nine different types of viruses can be detected by using an array of nine specifically corresponding types of antibodies. When the array is exposed to a target solution containing type-4 viruses, for instance, the viruses will specifically bind to antibodies in array point 4. This binding can be detected using the CMOS IC below in much the same ways DNA hybridization is detected. By reading the location of the binding, the presence and type of viruses are determined. The multiplexed array platform can be especially useful for the diagnostics of complex diseases like cancer.

A new threat

Imagine this: at the Olympic games in London in 2012, a little known athlete surprises everyone in the men’s 100 metres, thrashing the established favourite by almost a whole second. “Is he on drugs?” everyone asks. Tests find nothing, and he is labelled a freak of nature. But are his achievements thanks to natural ability or are they down to genetic modification?

In our darkest memories, we can all recall having lied, cheated, or stretched the rules, often to gain a personal advantage. For elite athletes the pressures that drive cheating are vastly increased. Sport requires that participants train hard over long periods, often from a young age, linking achievement to self esteem, emotional state, and lifestyle. The temptations of money and fame and pressure from parents, coaches, and fans drive athletes to accept extreme risk to gain even small advantages.

Perhaps it is unsurprising that some athletes turn to science and drugs to improve their chances. In 1967 the British cyclist Tom Simpson collapsed and died on the slopes of Mont Ventoux, in France, largely because of misuse of amphetamines. As tests were developed a number of high profile cases brought doping into the public eye: Ben Johnson was stripped of his gold medal in the 1988 Olympic games, and the whole Festina team was ejected from the Tour de France in 1998 after doping equipment was found in the car of a team official.

An independent international body was established to fight doping on a global scale—the World Anti-Doping Agency. It updates the world antidoping code, which contains a list of prohibited substances and practices; coordinates the testing and education of athletes and their coaches; and funds research into testing to remain one step ahead of potential cheats.

In 2002 the agency recognised a new threat—“gene doping”—by including it in the list of prohibited practices, defined as “the non-therapeutic use of cells, genes, genetic elements, or of the modulation of gene expression, having the capacity to enhance athletic performance.

The techniques involved in gene doping are spawned from the techniques of gene therapy, in particular gene transfer with viral vectors (fig 1). The aim of gene manipulation in these two settings can be clearly distinguished. In gene therapy the goal is treatment and requires the replacement of a defective gene or expression of a therapeutic gene. Gene doping, however, aims to improve athletic performance by increasing or decreasing production of endogenous molecules.