31 March 2008

Nanomachines will signal about single DNA molecules

Scientists at Arizona State University, working under the leadership of Dr. Wayne Frasch, have developed a biosensor nanodetector, which in the future may lead to a revolution in the field of medical screening for various diseases and (which is especially important for obtaining government funding :) it will allow you to instantly identify bioterrorists – for example, at airports.

An experimental biosensor can be compared to a mass of microscopic beacons that flash upon contact with a certain sequence of nucleotides characteristic of the DNA of only a certain virus, bacterium or oncogene. At the same time, the sensitivity of the new sensor is millions of times higher than that of existing DNA detection methods.

The basis of the "beacon" is F1-adenosine triphosphatase (F1-ATPase), which belongs to a group of enzymes that provide energy synthesis in all organisms, including the process of photosynthesis in plant cells. For studying the structure and characteristics of F1-ATPase, two Nobel Prizes were awarded – in 1979 and 1997. This enzyme cleaves adenosine triphosphate (ATP) to adenosine diphosphate (ADP) with the release of energy. The diameter of the enzyme molecule is 10-12 nm, and it works about the same as a three-cylinder rotary engine of Mazda, rotating a protein "rotor" protruding from a "stator" consisting of six globules. F1-ATPase molecules are applied to the substrate at a distance of about 1 micron from each other. In the macrocosm, it would look like engines about a meter in size, placed at a distance of 100 meters from each other in the boundless steppe: a centimeter with such an increase will turn into a thousand kilometers. The developers attached a molecule of avidin protein to each "rotor" (why – it will be clear from the further description).

Gold nanorods about 90 nm in length and 35 in diameter are used as "lamps", when rotating, flashes of reflected light are visible in the microscope. The nanorods are also coated with avidin molecules.

The third element of the system is a single–stranded DNA molecule, complementary to the target – to be detected DNA sequence, 40 nucleotides long. The ends of the sensory DNA chain are biotinylated – connected to biotin molecules. Biotin (vitamin H) is able to bind strongly to avidin (avis is Latin for bird, and both substances were first isolated from chicken eggs).

And finally, in order for the "cardan shaft" of the nanodetector to become sufficiently rigid, it is necessary to add a second DNA chain to it. The sample (with a volume of 3 microliters) is heated almost to a boil. At the same time, the double DNA strands unravel, and when cooled to room temperature, they connect again – both with each other and with the sensory sequence of nucleotides (with the latter, of course, only if they are complementary to each other).

When the "fuel" – ATP – is added to the solution, those of the nanorods that are connected to the ATPase molecules by double DNA chains begin to rotate. How it looks under the microscope, you can see on the video (avi, 3.5 MB). The spectacle, however, is not exciting: well, flashing lights on a black background…

The analysis takes only 15 minutes – an order of magnitude less than using existing methods, using first polymerase chain reaction (PCR) to multiply the number of DNA molecules multiple times, and then immunofluorescence to detect a certain sequence of nucleotides in the sample.

To carry out enzyme immunoassay, the DNA concentration must be increased to at least 5 picomoles (picomoles – 10-12 moles); using plasmon resonance and Raman spectroscopy methods used for the same purpose, DNA can be detected at a concentration of 10-20 femtomoles (femto - - 10-15). The developers of the new method jumped the attomolar boundary (atto- – 10-18): using the nanosensor described above, it is possible to detect zeptomolar DNA concentrations (zeptomol – 10-21 moles, total 600 molecules). Further along this scale is the yoctomol, but purely formally: this is already 0.6 molecules. And an advanced ATPase biosensor will allow you to quickly and accurately identify literally single DNA molecules.

By the way, the description of the nanosensor and the details of its work in our retelling is pretty simplified. You can read more about this in the original article by Justin York et al. “Single-molecule detection of DNA via sequence-specific links between F1-ATPase motors and gold nanorod sensors” (Lab Chip, 2008, 8, 415-419, DOI: 10.1039/b716744j). Unfortunately, it is not publicly available.

The authors count on the support of the Arizona Science Foundation in transferring the results of their work from the laboratory to practical biotechnology by creating a company specializing in the use of F1-ATPase in the production of DNA detection devices.

A prototype of such a device is already being developed. To use it, a smear from an infected wound or baggage should be dissolved in a special composition, a drop of which is applied to a slide to which reference F1-ATPases with nanorods are attached. At the same time, a flashing light signal recorded by a computer indicates the presence of the desired DNA in the smear.

The authors have already received funding to develop a similar method for detecting proteins at the level of single molecules. This direction is particularly promising, because, unlike DNA, proteins cannot be amplified in the laboratory to increase the chances of detection.

Evgeniya Ryabtseva
Portal "Eternal youth" www.vechnayamolodost.ru based on the materials of ScienceDaily


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