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November/December 2009
Feature

Putting It to the Test

ILS Program Supports Precision and Bias Statement Development for Nanotechnology Standard

In a field where size is everything, the ability to accurately measure size is very important.

That’s the case with nanotechnology. And the recent revision of E2490, Guide for Measurement of Particle Size Distribution of Nanomaterials in Suspension by Photon Correlation Spectroscopy, contains a precision and bias statement to check that accuracy. Extensive testing by independent laboratories working under the guidance of the ASTM International Interlaboratory Study Program has led to the E2490 statement.

The ILS Program

The ILS Program helps ensure that every ASTM International test method is validated by a precision statement outlining what users can expect from a specific protocol in terms of repeatability (i.e., what one laboratory analyzing the same sample multiple times would see as its range of acceptable results) and reproducibility (i.e., the differences that would be expected if the same sample were analyzed using the same method in multiple laboratories).

“Without these studies, there is no basis for judging the accuracy or precision of a method or standard,” says Vincent Hackley, Ph.D., leader of the Nanoparticle Metrology and Standards for Biomedical Applications and Health project at the National Institute of Standards and Technology, Gaithersburg, Md. “They are necessary to lay the groundwork for future industry standards and to validate new standards as they appear.”

Precision statements supported by data-driven studies enable users, producers and consumers to have greater confidence in the ASTM International test methods that they reference. The ILS Program helps provide that added confidence by relieving committees of the administrative duties associated with the development of precision and bias statements — a process that can be difficult, time consuming and expensive.

Participation in the program is voluntary; committees are not required to use ILS services, but about half of ASTM’s technical committees have or are currently taking advantage of the program.

“This is a competitive advantage that other organizations don’t have,” says Alan Rawle, Ph.D., applications manager, Malvern Instruments Inc., Westborough, Mass., and co-chair of Subcommittee E56.02 on Characterization: Physical, Chemical and Toxicological Properties, part of Committee E56 on Nanotechnology. “Other organizations basically rely on the efforts of their volunteers, as ASTM does, but the ILS studies are vital in giving credence to a method or methodology.”

The program was originally developed as a five-year pilot but due to the amount of positive feedback from past and current participants, the ILS program was recently approved as a permanent part of the ASTM budget.

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The E2490 ILS program and two related studies utilized gold nanoparticle reference materials from NIST.

How Small is Small?

Nanotechnology is the science of very small things, but how small is small?

According to the National Nanotechnology Initiative, nanotechnology is defined as “the understanding and control of matter at dimensions between approximately 1 and 100 nanometers.” To put that in size perspective, a single molecule of water measures less than 1 nm — one-billionth of a meter — in length, and a human hair is about 100,000 nm wide.

“This is science and engineering at a scale comparable to the size of many biological components such as viruses, proteins and DNA,” explains Hackley. “We’re talking about the development of materials thousands of times smaller than the diameter of a human hair.”

E2490 and PCS

The revised standard, E2490, deals with the measurement of nanoparticles using a method called photon correlation spectroscopy. Also known as dynamic light scattering, PCS is one of the few widely available methods capable of providing accurate nanosize measurements of particles dispersed in liquid media, such as in buffer solutions frequently utilized for in vitro testing. It is also one of the most commonly used methods due to its ease of operation, low entry costs and minimal preparation requirements.

“The technique works because of the special properties of nanoparticles when suspended in liquid,” explains Anil Patri, Ph.D., deputy director of the National Cancer Institute’s Nanotechnology Characterization Laboratory, Frederick, Md. When a laser is focused through the sample to be measured, the light is deflected or “scattered” in different directions depending on the size and motion of the particles in suspension.

“Nanoparticles have a random motion in liquid,” says Patri. “Their movement is related to their size, so with the proper instruments we can observe their movement and the light scatter, and calculate the size or range of sizes of the nanoparticles. Different environmental factors such as temperature of the medium, the viscosity of the liquid, etc., can affect the way the particles move, so having precise testing protocols is extremely important.”

The ILS Program for E2490

ILS Study 166 on the physical properties of nanoparticles provided the data for E2490. Twenty-six laboratories took part in the study on the size characteristics of nanoparticles. Although primarily using the PCS method, the study also included corollary microscopy-based techniques and allowed the involved groups to compare their performance against other laboratories and against the stated results from NIST.

The end of study report noted that the testing yielded very favorable results, and the data accumulated by the test labs was close to the data expected by NIST. Although the test values were a little higher than the NIST values, they were still within an acceptable range.

“We had well-defined reference materials so we went into it knowing what the end results should be,” Hackley says. “The goal was not to test the material itself, but the methodology, the process, to see how much variability occurred between labs using the same procedures.”

“This was the first nanotechnology standard published that had an interlab study associated with it,” says Martin Fritts, Ph.D., senior principal scientist, NCL. “It was important to put out a protocol that not only examined how nanomaterials should be measured but also to examine the uncertainties and bias inherent in executing the same method over many different labs.”

Determining the level of uncertainty was a key part of the study, notes Patri. “That information can be incorporated into regulation for researchers and manufacturers. We were very happy with the outcome. The testing was very thorough and showed very little variation.”

Additional Testing

In addition to ILS 166, two other nanotechnology-related studies were performed. ILS 201 and 202 were conducted to examine the biological properties of nanomaterials — specifically, their effect on human blood and other live cells. Although the testing phase of these studies was ruled “inconclusive,” researchers believe that they collected valuable information about the planning and management of protocols and supplies.

“These studies illustrated the difficulties of working with living things such as human blood and cells in comparison with the ease of working with inanimate objects and equipment,” says Rawle. “Any technique has potential difficulties or problems associated with it, and studies like these help expose those difficulties.”

ILS 201 and 202 served as guides to dealing with everything from the difficulty of transporting samples during a New England winter — some samples arrived at their destinations frozen and unusable — to how many decimal points are realistic to use in nanoparticle measurement.

“What we learned is that biological studies need to be more carefully and rigorously defined in terms of methods, methodologies and data gathering,” says Rawle. “It’s like trying to design a diet program for the entire population — it just isn’t going to work. You need to tailor your approach to each individual.”

Synergy

These ILS studies represent a huge undertaking, and a number of factors came together to help make them possible. In addition to ASTM International, groups like NIST, the National Cancer Institute and its Nanotechnology Characterization Laboratory played key roles in making these tests a reality. NIST especially had a major role, providing different size gold nanoparticle reference materials (RMs 8011, 8012 and 8013 were developed in their labs with funding in part from NCI) for use as test materials. Additional test materials included commercially acquired dendrimers (nanosized symmetrical molecules with tree branch-like structures).

Hackley says, “It is the confluence of reference material availability, development of ASTM nanotechnology standards, interagency cooperation and the ILS program support from ASTM that made these studies possible and successful.”

Looking Ahead: Uses of Nanotechnology

At present there are more than 800 products on the market that make use of nanotechnology. Eddie Bauer and Dockers brands feature clothing lines treated with nanofibers to increase stain resistance. Automobile makers General Motors Co. and Toyota Motor Corp. use nanotechnology to produce materials that are lighter, rustproof and scratch resistant. Golf clubs and tennis rackets made by Wilson Sporting Goods Co. are lighter and stronger thanks to the use of nanomaterials in their construction.

Of great interest to scientists is how nanotechnology will be applied to medical research, especially in the area of cancer treatment.

“Nanoparticles can be used in biomedical applications as vehicles to carry a payload — therapeutic drugs or imaging agents — to a targeted site in the body,” notes Hackley, who is also a principal investigator in NIST’s collaboration with the NCL. “The particles are small enough, and can be made sufficiently invisible to the immune system, that they can circulate in the bloodstream and cross cellular and other biological barriers to reach a specific destination.”

Targeting a specific destination in the body can be accomplished by creating the right sized particle. For example, nanomaterial with a size of less than 10 nm injected into the body would be eliminated through the kidneys. Particles with a size of 30 nm would be too large to be expelled by the kidneys but may be expelled by the liver instead.

“For cancer treatments,” says Hackley, “nanotechnology may provide more sensitive detection and location of tumors, less systemic damage from anti-cancer drugs or radiation treatments, or combined imaging and therapeutics.”

E2490 and other new standards will be used by scientists in the pharmaceutical, cancer research and nanotechnology fields as well as regulatory and environmental health and safety agencies, according to Fritts. “Standardization should accelerate the translation of nanotech therapeutics from the proof-of-concept discovery phase into clinical trials and eventually into commercially available drugs and benefits to patients,” he says.

Additionally, standards will help address concerns regarding the potential harmful effects of engineered nanomaterials on human health and the environment. Validated measurements backed by interlaboratory testing will be critical in ensuring that these concerns are addressed in a systematic and reproducible manner.

For More Information

If you would like to know more about the ILS Program, contact Phillip Godorov, ILS program director, ASTM International (phone: 610-832-9715). For more information about Committee E56, contact Timothy Brooke, Technical Committee Operations, ASTM International (phone: 610-832-9729).

 

Kessel Nelson is a freelance writer whose work has appeared in national and international publications, covering subjects ranging from art to energy to schizophrenia. He has a B.A. in history from the University of Pennsylvania, and he spends his time between Philadelphia and New York City.