Nanotechnology Advances

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By Leo O’Connor, Katherine Austin and Michael Valenti

While working hard to develop practical nano-sized devices, researchers are confident that biotechnology will be a central driving force for nanotechnology’s first group of successful applications. Biomedical research, drug delivery, and disease diagnosis rate high on the list of nanotechnology discoveries expected to achieve commercial success.

The goal for nanotechnologists is to produce biosensors that have better sensitivity, specificity and stability, and decreased manufacturing costs over currently available equipment.

In a report recently released by Technical Insights (a division of San Antonio, Texas-based Frost & Sullivan), entitled Biosensors: Emerging Technologies and Growth Opportunities, nanotube researcher Richard Smalley, PhD reports on specific nanotechnology developments that may lead to commercial applications in biotechnology. He said, “Some of the most sophisticated biomedical tests today — such as MRI exams — cannot be performed in a doctor’s office because the equipment is too large and too expensive to operate. Because nothing in the human body fluoresces in the near-infrared spectrum, and human tissue is fairly transparent at that spectrum, one can envision a test apparatus based on nanotechnology that would be as inexpensive and simple to use as ultrasound.”

Smalley led a team at Rice University (Houston, TX) that saw the first fluorescence in carbon nanotubes. Fluorescence happens when a substance absorbs one wavelength of light and emits a different wavelength. The team found that nanotubes absorb and give off light in the near-infrared spectrum, which will be good for biomedical and nanoelectronics applications.

The tubes could also be used as optical biosensors that could be set up to seek out specific targets within the body such as tumour cells or inflamed tissues. The tubes would be wrapped with protein that would bind only to the target cells. Since nanotubes fluoresce with a single wavelength of light and different-diameter nanotubes give off different wavelengths, it should be possible to tailor different sizes of tubes to seek specific targets. This would be a way to diagnose multiple maladies in a single test using a cocktail of nanotubes.

Fluorescence from Nanotubes

Nanotubes are direct band-gap semiconductors, which means they emit light in an interesting way.

The Rice team observed fluorescence directly across the band gap of semiconducting carbon nanotubes. Individual nanotubes, each encased in a cylindrical micelle, were created by ultrasonically agitating an aqueous dispersion of raw single-walled carbon nanotubes in sodium dodecyl sulfate, then centrifuging to remove tube bundles, ropes, and residual catalysts.

When nanotubes are aggregated into bundles, the fluorescence is generally quenched through interactions with metallic tubes, substantially broadening the absorption spectra. At a pH of less than five, absorption and emission spectra of individual nanotubes show evidence of band-gap-selective protonation of the tubes’ sidewalls. This protonation is readily reversed by treatment with base or ultraviolet light.

Getting single tubes out of the pack was a major accomplishment. Preparation methods produce a diversity of tube diameters, chiral angles and aggregation states. Aggregation is a problem because the highly polarizable, smooth-sided fullerene tubes readily form parallel bundles or ropes with van der Waals binding energy of 500 eV per micrometre of tube-to-tube contact.

The Rice team devised a method that detaches tubes from bundles by physical means, applies a non-perturbing coating to prevent re-aggregation, then removes any remaining bundles from the solution.

Smalley, working with fellow chemist R. Bruce Weisman, PhD, is now trying to bridge the gap between wet nanotechnology (the molecular, biochemical machinery of life) and dry nanotechnology (insoluble nanomaterials such as fullerenes). Dozens of varieties of soluble fullerenes have been produced by wrapping nanotubes in various polymers, including proteins, starches and DNA. Fluorescence was seen in both plain and polymer-wrapped nanotubes.

Market Promise

Although much of the work in nanotechnology is still confined to laboratories, a program established by the Advanced Technology Program (ATP) division of the National Institute of Standards and Technology is designed to more rapidly move chemical sensor and biosensor technologies from the research laboratory toward the marketplace, by supporting programs that improve sensors and sensor integration. According to the ATP, these improvements will permit such sensors to become more competitive and increase their penetration into medical/clinical diagnostics, bioprocess monitoring, food and beverage monitoring, and environmental monitoring markets. Biosensors and chemical sensors currently in the marketplace are sold only into niche markets because they cannot meet the criteria that would allow them to compete more widely.

According to the ATP, current commercial expectations in the chemical sensor and biosensor fields remain essentially flat at 3% to 7% growth through 2003. Only the medical research area is expected to grow at 10% to 14% through the same period. The hope of the ATP Sensor Program is that, with the introduction of advanced technologies and manufacturing, the biosensor yearly sales in all areas can be improved by as much as four-fold per year.

The ATP estimates that if its program in nano and MEMS (microelectro-mechanical systems) technologies for chemical biosensors focuses on cutting-edge technologies employing nanofabrication and microfabrication that provide integrated sensor arrays, ultra-high sensitivities, ease of use, reliability, and real-time measurements, such technological advances should increase the chemical sensor and biosensor market share; increase the size of the U.S. sensor manufacturers; and increase the movement of sensor technology into new markets. This would result in the creation of a combined market for chemical sensors and biosensors of over $4 billion US by 2007.

Nanochips

Detection of micro-organisms is an increasing concern and researchers just don’t have the right tools to seek them out in all the places they lurk. Food and water safety, biological warfare and disease diagnosis all need a simple, rapid and easy microbiological test. Researchers at Nanogen Inc. (San Diego, CA) have developed a bioelectronic chip that isolates, lyses and detects micro-organisms in complex samples. Nanogen’s NanoChip® contains an addressable array of 25 platinum 80-µm electrodes.

Each chip is covered by an agarose permeation layer in a 4.84 µl flow cell. The chip is manufactured by chip fabrication techniques. The alternating current field setup in the chip separates cells by dielectrophoresis. Different cell types move to different regions of the array. Weakly held cells located in interelectrode regions of low field strength can be selectively removed by washing. Escherichia coli cells can be isolated from a whole blood sample in about four minutes. A series of 500 V pulses lyses the E. coli onto the microelectrode array, which is then incubated with proteinase-K to digest proteins.

The lysate is moved to another bioelectronic chip for an electronically enhanced hybridization assay. The microfabricated device effectively integrates the often complicated sample preparation steps on a one- to two-centimetre analytical chip. Dielectrophoretic separation of E. coli from the blood cells, electronic lysis of the isolated E. coli, and digestion of proteins are performed on one chip contained in a flow chamber. The same chip has been used to separate Micrococcus lysodeikticus, Staphylococcus epidermidis, and cultured cervical carcinoma cells from whole human blood. Sample preparation has proven to be the hardest part of trying to put a lab on a chip.

Nanogen is working with the support of a three-year, $1.5-million US grant from the U.S. Army to continue development of miniaturized electronic devices for isolating and detecting biological warfare and infectious disease agents from human blood samples.

DNA Analysis

An integrated device developed by University of Michigan (Ann Arbor, MI) researchers crams everything needed for conventional biochemical DNA analyses onto a single glass and silicon chip that could eventually be incorporated into portable instruments for medical, forensic and agricultural diagnostics. According to a report in Science, the Michigan group, led by Mark Burns, PhD, has devised a lab-on-a-chip that analyses nanolitre-size DNA samples. The chip measures reagent and DNA solutions, mixes them together, amplifies or digests the DNA into products, then separates and detects those products.

The system includes a nanolitre liquid injector, a sample mixing and positioning system, a temperature-controlled reaction chamber, an electrophoretic separation system and a fluorescence detector — the first time such a detector has been integrated onto a DNA analysis chip. DNA and reagent solutions are placed on the device and electronic signals corresponding to genetic information are the primary output. Using a pipette, a sample of DNA-containing solution is placed on one fluid-entry port and a reagent-containing solution on the other port. Capillary action draws both solutions into the device, but hydrophobic patches positioned just beyond the vent line in each injection channel stop the samples. Pressure through an air vent pinches off precise nanolitre drops, which mix together and move forward to the thermal reaction chamber. Microheaters and temperature sensors control the temperature in the reaction chamber for a specified time. The device also detects size-fractionated products with microfabricated fluorescence detectors.

The output from the detector indicates the migration time of the DNA reaction products. Burns’s team is currently trying to improve the resolution of the separation system and increase the number of reaction/separation components on the device. His group has applied for patents and is negotiating with several companies. One consortium that recently received an Advanced Technology Program award to develop a DNA lab-on-a-chip should be particularly eager to talk with Burns. Applied Biosystems (Foster City, CA) is leading a group developing an integrated set of microdevices for automated separation and purification of DNA. These lab-on-a-chip devices are being designed to perform all steps from sample input to purification needed to ready samples for several different analytical techniques. The preliminary estimate on the cost of producing the device in research-sized quantities is approximately $6 US per unit. However, mass production would lower that cost.

This article was compiled by Leo O’Connor, global director of Research with Technical Insights, a division of Frost & Sullivan (San Antonio, TX). It includes reporting and analysis by Katherine Austin and Michael Valenti, who are both members of the Technical Insights information research team.