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Topic Name: Arizona Researcher Developed Biosensing Nanodevice that can Revolutionize Health Screenings

Category: Biodesign

Research persons: Wayne Frasch

Location: Arizona State University, United States

Details

One day soon a biosensing nanodevice developed by Arizona State University researcher Wayne Frasch may eliminate long lines at airport security checkpoints and revolutionize health screenings for diseases like anthrax, cancer and antibiotic resistant Staphylococcus aureus (MRSA).

Even more incredible than the device itself, is that it is based on the world’s tiniest rotary motor: a biological engine measured on the order of molecules.

Frasch works with the enzyme F1-adenosine triphosphatase, better known as F1- ATPase. This enzyme, only 10 to 12 nanometers in diameter, has an axle that spins and produces torque. This tiny wonder is part of a complex of proteins key to creating energy in all living things, including photosynthesis in plants. F1-ATPase breaks down adenosine triphosphate (ATP) to adenosine diphospahte (ADP), releasing energy. Previous studies of its structure and characteristics have been the source of two Nobel Prizes awarded in 1979 and 1997.

It was through his own detailed study of the rotational mechanism of the F1-ATPase, which operates like a three-cylinder Mazda rotary motor, that Frasch conceived of a way to take this tiny biological powerhouse and couple it with science applications outside of the human body.

An article authored by Frasch and his colleagues in the ASU School of Life Sciences details the technology that would allow this. Their publication “Single-molecule detection of DNA via sequence-specific links between F1-ATPase motors and gold nanorod sensors” was recently published in the journal Lab on a Chip, and featured in the online journal Chemical Biology produced by the Royal Society of Chemistry.

What Frasch and his colleagues show is that the enzyme can be armed with an optical probe (gold nanorod) and manipulated to emit a signal when it detects a single molecule of target DNA. This is achieved by anchoring a quiescent F1-ATPase motor to a surface. A single strand of a reference biotinylated DNA molecule is then attached to its axle. The marker protein, biotin, on the DNA is known to bind specifically and tightly to the glycoprotein avidin, so an avidin-coated gold nanorod is then added. The avidin-nanorod attaches to the biotinylated DNA strand and forms a stable complex.

When a test solution containing a target piece of DNA is added, this DNA binds to the single complementary reference strand attached to the F1-ATPase. The DNA complex, suspended between the nanorod and the axle, forms a stiff bridge. Once ATP is added to the test solution, the F1-ATPase axle spins, and with it, the attached (now double-stranded) DNA and nanorod. The whirling nano-sized device emits a pulsing red signal that can then be detected with a microscope.

According to Frasch, the rotation discriminates fully assembled nanodevices from nonspecifically bound nanorods, resulting in a sensitivity limit of one zeptomole (600 molecules). Simply put, if it’s not moving and flashing, it simply isn’t relevant.

Moreover, Frasch says, “Studies with the F1-ATPase in my laboratory show that since it can detect single DNA molecules, it far exceeds the detection limits of conventional PCR [polymerase chain reaction] technology.”

Such a detection instrument based on the F1-ATPase enzyme would also be “faster and more portable,” he adds.

With support from Science Foundation Arizona (SFAz), Frasch will transfer his work from the bench to biotech, through establishment of a local company that utilizes the nano-sized F1-ATPase to produce a DNA detection instrument.

A prototype of the DNA detector is already in development. It is roughly the size of a small tissue box. Sampling would be as simple as taking a swab from an infected wound or a piece of baggage, dissolving it in a solution and placing a drop on a slide bearing reference F1-ATPases and their nanorods. Once in the instrument, red blinking signals emitted by rotating nanorods would let a computer know there’s trouble, literally, in a flash.

SFAz funding has also enabled Frasch to extend the method to do protein detection at the single molecule level. This is novel because, unlike DNA, proteins can not be amplified artificially to improve the chances of detection.

“Rapid and sensitive biosensing of nucleic acids and proteins is vital for the identification of pathogenic agents of biomedical and bioterrorist importance,” notes Frasch, who is also with the Center for Bioenergy and Photosynthesis in the College of Liberal Arts and Sciences. “It also provides a new avenue through which to analyze genotypes and forensic evidence.”

Note for Anthrax
Anthrax is an acute disease in humans and animals that is caused by the bacterium Bacillus anthracis and is highly lethal in some forms. Anthrax is one of only a few bacteria that can form long-lived spores. When the bacteria’s life cycle is threatened by factors such as lack of food caused by their host dying or by a change of temperature, the bacteria turn themselves into more or less dormant spores to wait for another host to continue their life cycle.

After ingesting or getting spores in a cut in the skin, a new host allows these spores to reactivate themselves and multiply in their new host very rapidly. The anthrax spores in soil are very tough and can live many decades and perhaps centuries and are known to occur on all continents except Antarctica. Anthrax most commonly occurs in wild and domestic grass-eating mammals (ruminants) who ingest or breathe in the spores while eating grass. Anthrax can also be caught by humans when they are exposed to dead infected pigs, eat tissue from infected animals, or are exposed to a high density of anthrax spores from an animal's fur, hide, or wool. Anthrax spores can be grown outside the body and used as a biological weapon. Anthrax cannot spread directly from human to human; but anthrax spores can be transported by human clothing, shoes etc. and if a person dies of anthrax their body can be a very dangerous source of anthrax spores. The word anthrax is the Greek word for coal, the germ's name is derived from anthrakitis, the Greek word for anthracite, in reference to the black skin lesions victims develop in a cutaneous skin infection.

Anthrax is one of the oldest recorded diseases of grazing animals such as sheep and cattle and is believed to be the Sixth Plague mentioned in the Book of Exodus in the Bible. Anthrax is also mentioned by Greek and Roman authors such as Homer (in The Iliad), Virgil (Georgics), and Hippocrates. Anthrax can also infect humans, usually as the result of coming into contact with infected animal hides, fur, wool ("Woolsorter's disease"), leather or contaminated soil. Anthrax ("siberian ulcer") is now fairly rare (a few to no cases per year in the developed world) in humans although it still occasionally occurs in ruminants, such as cattle, sheep, goats, camels, wild buffalo, and antelopes.

Bacillus anthracis bacteria spores are soil-borne and because of their long lifetime they are still present globally and at animal burial sites of anthrax-killed animals for many decades; spores have been known to have reinfected animals over 70 years after burial sites of anthrax-infected animals were disturbed.

Anthrax was first known to infect animals in the year 17 B.C. when a French farmer noticed his sheep flock suddenly dying. Bacillus Anthracis is a rod-shaped Gram-positive bacterium, about 1 by 9 micrometers in size. It was shown to cause disease by Robert Koch in 1877. The bacterium normally rests in endospore form in the soil, and can survive for decades in this state. Ruminants are often infected whilst grazing, especially when grazing rough, irritant or spiky vegetation: the vegetation causes wounds within the gastrointestinal tract permitting entry of the bacterial endo-spores into the tissues. Once ingested by a ruminant or placed in an open cut, the bacterium begins multiplying inside the animal or human and in a few days to a month kills it. The endo-spores germinate at the site of entry into the tissues and then spread via the circulation to the lymphatics, where the bacteria multiply. It is the production of a powerful exo-toxin by the bacteria that causes death. Veterinarians can often tell a possible anthrax-induced death by its sudden occurrence and by the blood and bloody fluids that oozes from the body orifices. Most anthrax bacteria inside the body are destroyed by anaerobic bacteria that can grow without oxygen. The greater danger lies in the bodily fluids and blood that spills from the body and spill into the soil where the anthrax bacteria turn into a dormant protective spore form. Once formed the spores are very hard to eradicate.

Note for Methicillin-Resistant Staphylococcus Aureus
Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium responsible for difficult-to-treat infections in humans. It may also be referred to as multiple-resistant Staphylococcus aureus or oxacillin-resistant Staphylococcus aureus (ORSA). The organism is often sub-categorized as Community-Associated MRSA (CA-MRSA) or Hospital-Associated MRSA (HA-MRSA) depending upon the circumstances of acquiring disease, based on current data that these are distinct strains of the bacterial species.

MRSA is a resistant variation of the common bacterium Staphylococcus aureus. It has evolved an ability to survive treatment with beta-lactam antibiotics, including penicillin, methicillin, and cephalosporins. MRSA is especially troublesome in hospital-associated (nosocomial) infections. In hospitals, patients with open wounds, invasive devices, and weakened immune systems are at greater risk for infection than the general public. Hospital staff who do not follow proper sanitary procedures may transfer bacteria from patient to patient.

MRSA/Multidrug Resistant Staphylococcus aureus was discovered in 1961 in the UK. It is now found worldwide. MRSA is often referred to in the press as a "superbug."

In the past decade or so the number of MRSA infections in the United States has increased significantly. A 2007 report in Emerging Infectious Diseases, a publication of the Centers for Disease Control and Prevention (CDC), estimated that the number of MRSA infections treated in hospitals doubled nationwide, from approximately 127,000 in 1999 to 278,000 in 2005, while at the same time deaths increased from 11,000 to more than 17,000. Another study led by the CDC and published in the October 17, 2007 issue of the Journal of the American Medical Association estimated that MRSA would have been responsible for 94,360 serious infections and associated with 18,650 hospital stay-related deaths in the United States in 2005. These figures suggest that MRSA infections are responsible for more deaths in the U.S. each year than AIDS.

The UK Office for National Statistics reported 1,629 MRSA-related deaths in England and Wales during 2005, indicating a MRSA-related mortality rate half the rate of that in the United States for 2005, even though the figures from the British source were explained to be high because of "improved levels of reporting, possibly brought about by the continued high public profile of the disease" during the time of the 2005 United Kingdom General Election.

It has been argued that the observed increased mortality among MRSA-infected patients may be the result of the increased underlying morbidity of these patients. Several studies, however, including one by Blot and colleagues, that have adjusted for underlying disease still found MRSA bacteremia to have a higher attributable mortality than methicillin-susceptible Staphylococcus aureus (MSSA) bacteremia.

While the statistics suggest a national epidemic growing out of control, it has been difficult to quantify the degree of morbidity and mortality attributable to MRSA. A 2004 study showed that patients in the United States with S. aureus infection had, on average, three times the length of hospital stay (14.3 vs. 4.5 days), incurred three times the total cost ($48,824 vs $14,141), and experienced five times the risk of in-hospital death (11.2% vs 2.3%) than patients without this infection. In a meta-analysis of 31 studies, Cosgrove et al, concluded that MRSA bacteremia is associated with increased mortality as compared with MSSA bacteremia (odds ratio = 1.93; 95% CI = 1.93±0.39). In addition, Wyllie et al. report a death rate of 34% within 30 days among patients infected with MRSA, a rate similar to the death rate of 27% seen among MSSA-infected patients.

Note for Adenosine 5'-triphosphate
Adenosine 5'-triphosphate (ATP) is a multifunctional nucleotide that is most important as a "molecular currency" of intracellular energy transfer. In this role, ATP transports chemical energy within cells for metabolism. It is produced as an energy source during the processes of photosynthesis and cellular respiration and consumed by many enzymes and a multitude of cellular processes including biosynthetic reactions, motility and cell division. In signal transduction pathways, ATP is used as a substrate by kinases that phosphorylate proteins and lipids, as well as by adenylate cyclase, which uses ATP to produce the second messenger molecule cyclic AMP.

The structure of this molecule consists of a purine base (adenine) attached to the 1' carbon atom of a pentose sugar (ribose). Three phosphate groups are attached at the 5' carbon atom of the pentose sugar. ATP is also incorporated into nucleic acids by polymerases in the processes of DNA replication and transcription. When ATP is used in DNA synthesis, the ribose sugar is first converted to deoxyribose by ribonucleotide reductase. ATP was discovered in 1929 by Karl Lohmann, and was proposed to be the main energy-transfer molecule in the cell by Fritz Albert Lipmann in 1941.

ATP consists of adenosine — itself composed of an adenine ring and a ribose sugar — and three phosphate groups (triphosphate). The phosphoryl groups, starting with the group closest to the ribose, are referred to as the alpha (α), beta (β), and gamma (γ) phosphates. ATP is highly soluble in water and is quite stable in solutions between pH 6.8–7.4, but is rapidly hydrolysed at extreme pH. Consequently, ATP is best stored as an anhydrous salt.

ATP is an unstable molecule and tends to be hydrolysed in water. If ATP and ADP are in chemical equilibrium, almost all the ATP will be converted to ADP. Any system that is far from equilibrium contains potential energy, and is capable of doing work. Biological cells maintain the ratio of ATP to ADP at a point ten orders of magnitude from equilibrium, with ATP concentrations a thousandfold higher than the concentration of ADP. This displacement from equilibrium means that the hydrolysis of ATP in the cell releases a great amount of energy. ATP is commonly referred to as a "high energy molecule"; however this is incorrect, as a mixture of ATP and ADP at equilibrium in water can do no useful work at all. ATP does not contain "high-energy bonds", and any other unstable molecule would serve equally well as a way of storing energy, if the cell maintained its concentration far from equilibrium.

The ATP concentration inside the cell is typically 1 - 10 mM. ATP can be produced by redox reactions using simple and complex sugars (carbohydrates) or lipids as an energy source. For ATP to be synthesized from complex fuels, they first need to be broken down into their basic components. Carbohydrates are hydrolysed into simple sugars, such as glucose and fructose. Fats (triglycerides) are metabolised to give fatty acids and glycerol.

The overall process of oxidizing glucose to carbon dioxide is known as cellular respiration and can produce up to 36 molecules of ATP from a single molecule of glucose. ATP can be produced by a number of distinct cellular processes; the three main pathways used to generate energy in eukaryotic organisms are glycolysis and the citric acid cycle/oxidative phosphorylation , both components of cellular respiration; and beta-oxidation. The majority of this ATP production by a non-photosynthetic aerobic eukaryote takes place in the mitochondria, which can make up nearly 25% of the total volume of a typical cell.

Some proteins that bind ATP do so in a characteristic protein fold known as the Rossmann fold, which is a general nucleotide-binding structural domain that can also bind the cofactor NAD. The most common ATP-binding proteins, known as kinases, share a small number of common folds; the protein kinases, the largest kinase superfamily, all share common structural features specialized for ATP binding and phosphate transfer.

ATP in complexes with proteins generally requires the presence of a divalent cation, almost always magnesium, which binds to the ATP phosphate groups. The presence of magnesium greatly decreases the dissociation constant of ATP from its protein binding partner without affecting the ability of the enzyme to catalyze its reaction once the ATP has bound. The presence of magnesium ions can serve as a mechanism for kinase regulation.

Note for Polymerase Chain Reaction
The polymerase chain reaction (PCR) is a technique widely used in molecular biology. It derives its name from one of its key components, a DNA polymerase used to amplify a piece of DNA by in vitro enzymatic replication. As PCR progresses, the DNA thus generated is itself used as template for replication. This sets in motion a chain reaction in which the DNA template is exponentially amplified. With PCR it is possible to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of the DNA piece. PCR can be performed without restrictions on the form of DNA, and it can be extensively modified to perform a wide array of genetic manipulations.

Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus. This DNA polymerase enzymatically assembles a new DNA strand from DNA building blocks, the nucleotides, using single-stranded DNA as template and DNA oligonucleotides (also called DNA primers) required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined series of temperature steps. These thermal cycling steps are necessary to physically separate the strands (at very high temperatures) in a DNA double helix (DNA melting) used as template during DNA synthesis (at lower temperatures) by the DNA polymerase to selectively amplify the target DNA. The power and selectivity of PCR are primarily due to selecting primers that are highly complementary to the DNA region targeted for amplification, and to the thermal cycling conditions used.

Developed in 1983 by Kary Mullis, PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensics and paternity testing); and the detection and diagnosis of infectious diseases. In 1993 Mullis won the Nobel Prize for his work on PCR.

In practice, PCR can fail for various reasons, in part due to its sensitivity to contamination causing amplification of spurious DNA products. Because of this, a number of techniques and procedures have been developed for optimizing PCR conditions. Contamination with extraneous DNA is addressed with lab protocols and procedures that separate pre-PCR mixtures from potential DNA contaminants. This usually involves spatial separation of PCR-setup areas from areas for analysis or purification of PCR products, and thoroughly cleaning the work surface between reaction setups. Primer-design techniques are important in improving PCR product yield and in avoiding the formation of spurious products, and the usage of alternate buffer components or polymerase enzymes can help with amplification of long or otherwise problematic regions of DNA.