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Superconducting SQUID microscope makes immunoassays easier, faster and more sensitive, scientists at UC Berkeley and LBNL report
08 Feb 2001

By Robert Sanders, Media Relations

Berkeley - Using an exquisitely sensitive magnetic field detector, a team of physicists, chemists and biochemists at the University of California, Berkeley, and Lawrence Berkeley National Laboratory (LBNL) has created a very sensitive and fast immunoassay.

Immunoassays are widely used in medical laboratories and industry to detect small levels of bacteria, drugs and many kinds of proteins or chemicals. The new technique, which relies on a so-called SQUID microscope, overcomes some of the drawbacks of standard immunoassays while speeding up the process.

"This technique could let you do in an hour or in minutes what now takes a day," said John Clarke, professor of physics in the College of Letters & Science at UC Berkeley and a faculty senior scientist in the Materials Sciences Division at LBNL. "If this really works, we could get information in real time, so that hospitals could diagnose an illness at the bedside, or food processors could find out immediately whether there is any bacterial contamination."

Aside from medical uses, a SQUID microscope also could be critical in bioterrorism situations where it is crucial to know the biological or chemical agent as soon as possible.

The new development was reported in the Dec. 19, 2000, issue of the Proceedings of the National Academy of Sciences.

The microscope relies on a device called a SQUID, or Superconducting Quantum Interference Device. Pioneered by Clarke over the past 35 years, the SQUID is the most sensitive detector of changes in magnetic field, and among other applications has been used to measure minuscule magnetic fields from the brain and the heart.

The SQUID is made from a high-temperature superconducting material - yttrium-barium-copper oxide - that operates at about 77 Kelvin, or 196 degrees Celsius below the freezing point of water. Though the SQUID is very cold, it can be brought close to living samples to detect small magnetic fields from them.

Clarke and graduate students Yann R. Chemla and Helene L. Grossman used the SQUID microscope to detect magnetic fields from various nearby sources, in this case nanometer-sized magnetic particles linked by antibodies to biological targets. The research team also included chemist Ray Stevens, a former UC Berkeley faculty member now at The Scripps Research Institute in La Jolla, Calif.; Mark Alper, adjunct professor of biochemistry and molecular biology at UC Berkeley and deputy head of the Materials Sciences Division at LBNL; and UC Berkeley undergraduate student Yan Poon.

In their initial experiments they were able to detect as few as 30,000 magnetic particles, which, if each were attached to a single target, means that their limit is about 30,000 cells or proteins. The most sensitive enzyme-linked immunosorbent assays (ELISA) can detect no fewer than 100,000 labeled targets.

However, refinements now underway should improve the sensitivity and allow them to detect as few as 50 to 500 magnetic particles. Since some bacteria have thousands of attachment sites for a particular antibody, theoretically the SQUID microscope could detect a single bacterium.

"This is fast and simple enough that you could use it in a batch process, matching the versatility of existing immunoassay methods," Clarke said. He said that an array of samples could readily be scanned over the SQUID.

To obtain high sensitivity, some immunoassay techniques require that cells be cultured overnight or longer in order to obtain a sufficient number to show up in an immunoassay. The sensitivity of the SQUID microscope makes this step unnecessary, thus making it faster.

Also, some current immunoassays label cells or molecules by attaching fluorescent or radioactive tracers with the help of antibodies designed to adhere selectively to the target. When irradiated with UV light the fluorescently-labeled targets light up, while the radioactively-labeled targets expose a film.

These techniques require that unattached tracers be flushed away, however. The new technique eliminates this step because magnetic tracers attached to the target behave differently than unattached tracers.

"A big part of the appeal of this technique is that you can easily distinguish between labeled and unlabeled particles," said Chemla.

This is possible because the nanoparticles are superparamagnetic, which means that when they encounter a magnetic field they become magnetized, line up along the field lines, and remain that way for a short time after the magnetic field is switched off. The aligned particles produce a net magnetic field that is strong enough to be detected by a SQUID.

If the nanoparticles are not attached to a target, however, the field generated by the aligned nanoparticles lasts only a short time before the magnets randomize as they jostle around (a process called Brownian rotation) and cancel one another out. If the nanoparticles are attached to a target that is in turn immobilized on a surface, though, the magnets can't reorient themselves. Instead, the spins of the individual atoms in the nanoparticle - the source of its magnetic dipole moment - are free to reorient themselves, eventually canceling out the magnetic dipole of the nanoparticle. This process is called NŽel relaxation.

For their technique to work, the physicists chose magnetic particles with complementary properties: when unattached, they randomize by Brownian rotation in less than a thousandth of a second; when attached, however, they require about a second to randomize via NŽel relaxation. Thus, when the SQUID microscope measures the decaying signal for a second after the outside field is switched off, the magnetic signal comes solely from the attached particles.

Because the sample takes only one second to magnetize and one second to demagnetize, detection takes as little as two seconds, Alper said. Even counting preparation time, he is optimistic that the whole process can be reduced to a minute or less.

"This could be used in a wide variety of applications to detect almost anything you can make antibodies against," he said. In addition, this technique could be used with any "molecular recognition element" - a molecule that can bind specifically to a particular surface feature on another molecule. Thus, the range of detectable targets is very broad and not limited to those against which antibodies can be produced.

"These are preliminary results from a device that hasn't yet been optimized," Alper cautioned. "Nevertheless, this is a clear scientific demonstration that you can apply these very, very sensitive magnetometers to the detection of biological substances."

Clarke, Alper and the students now are working with Paul Alivisatos, professor of chemistry and faculty senior scientist at LBNL, to come up with improved nanoparticles, and with Carolyn Bertozzi, UC Berkeley associate professor of chemistry and a member of LBNL's Materials Sciences and Physical Biosciences Divisions, to improve methods of attaching them to molecular recognition elements.

The work was supported by grants through LBNL from the Division of Materials Sciences, Office of Basic Energy Sciences, U.S. Department of Energy. Clarke and Alper are among several hundred UC Berkeley researchers involved with the campus's Health Sciences Initiative, which draws scientists from a broad range of fields to tackle today's health problems.