New miniaturized lipid biosensor has promise of rapidly detecting biological agents
Ultrathin double layers of self-assembled fat molecules — resembling and acting much like soap bubbles — are at the heart of a new, dime-size biosensor being developed at Sandia by a team of researchers led by Bob Hughes (1744).
The biosensor has the promise of rapidly detecting a variety of biological agents, including viruses, anthrax, and other bacteria, in the field with the same sensitivity and specificity as standard laboratory procedures.
"This biosensor is part of Sandia’s biostrategy, driven in one context by our role in defending against the biothreat on the battlefield or at home," says Sandia VP 1000 Al Romig. "One aspect of this defense is prompt and accurate detection of the threat. Our biosensor work, such as what Bob and his team are doing, is directed at such detection. Again, our unique contribution is the application of microsystems, materials science, and information technology to solving a problem in biotechnology with national security relevance."
To this end Bob is adapting electrical impedance detection technology, which he has used for chemical sensors such as the chemiresistor, to biological sensing. Developing biosensors is a natural growth of Sandia’s chemiresistor program — coupling the exquisite sensitivity and selectivity of biological systems to the simple measurement of change in electrical resistance. Sensors with electrical detection, like chemiresistors, are integrated with other electronic components in a microsystem and typically operate at very low power.
The chemiresistor has a base of wirelike electrodes on a specially designed microfabricated circuit. In past experiments Bob focused on volatile organic compounds (VOCs), depositing thin polymer films that detect specific VOCs by absorption. When the VOC molecules appear, the polymer absorbs them, causing the polymer to swell. The swelling changes the electrical resistance that then is measured and recorded, providing information to determine VOC type and concentration present.
Similarly, Bob’s new sensor, the lipid chip, has a base of wirelike electronics. However, instead of using polymers as the sensing materials, the new sensor uses organic lipid bilayers — self-assembled double layers of lipid molecules.
"Scientists have studied lipids, fatlike molecules, for many years and have a good understanding of their characteristics," Bob says. "What is new is integrating them into a rugged biosensor that can detect biological agents."
Tricky work
Working with the lipid bilayers is tricky, Bob says. They are very fragile, like soap bubbles, and have a characteristic that sets them apart from other organic molecules — their dual oil/water solubility. One part of a lipid molecule is hydrophilic, water-soluble; another part is hydrophobic, oil-soluble. The hydrophilic/hydrophobic characteristics allow the lipids to line up spontaneously (self-assemble). This layer of lipids, about five nanometers thick, is assembled across the electrodes.
"One of the first things we had to do was deal with the fragile nature of lipid bilayers," Bob says. "We had to come up with a way to make them rugged enough to last through experiments and for use in the field."
Bob and his fellow researchers attacked the robustness dilemma by several methods. One uses a thin film of sol-gel to act as a scaffold for the bilayers. The sol-gel scaffold containing the lipid bilayers are placed on top of the electrodes.
Templated sol-gel films are very thin (less than one micron), but very durable — much like glass, but formed from a jellylike mass of water, alcohol, and metal oxides. The films used in the lipid bilayer sensor were provided by Hongyou Fan and Darren Dunphy in Jeff Brinker’s (1846) material science group.
Another method, developed by Darren Branch (1744), uses a hybrid bilayer in which the layer next to the metal electrode is actually not a lipid but an organic silane that attaches to the metal, but still supports the upper lipid monolayer.
‘Gated channels’
Another challenge was to create "channels" — pores formed by proteins in the lipid bilayers that could open and close repeatedly in response to the presence of a specific biological agent. The interaction of the agent with the ion channels in the lipid bilayers is key to the sensor. In the presence of the biological agent, the ion channels can change the electrical impedance of the bilayer by allowing the conduction of ions through the bilayer. In this way the type and concentration of the agent can be identified by a measurement of electrical resistance.
The ion conduction property of these channels, when inserted in lipid bilayers, closely mimics their activity in living cells. The basic structure of the cell wall is a lipid bilayer, although there are many other complex structures also found in the cell wall. Gated ion channel proteins in the cell walls are often involved in exquisitely sensitive chemical detection (often a single molecule can cause the opening or closing of an ion channel).
The study of membrane-bound proteins is an active area of research including protein structure and function in bilayer assemblies and the potential applications in biotechnology, medicine, and biosensing. Sandia is actively involved in this area of research in the Interfacial Bioscience (IBIG) Grand Challenge (Lab News, July 12).
The challenge facing biosensor developers is how to trigger the proteins to recognize when to "open" or "close" their channels in response to a target agent. The ion channels don’t work at all if they are not bathed in the relatively fluid lipid bilayer, so it is not possible to use them as sensors in rugged polymer matrixes like the chemiresistors for VOCs.
Use of antibodies
The solution will ultimately be to attach antibodies or other molecular recognition molecules to capture the desired molecules on the ion channels. Bob uses anthrax to explain.
"If, for example, you want to know if anthrax is present, you would attach anthrax antibodies to the ion channel protein in the bilayer," Bob says. "When an anthrax spore comes along, it would attach to the anthrax antibodies connected to the ion channel protein. The protein would change the bilayer’s electrical properties in response to the binding."
Bob says the research team has come a long way in the short time it’s been working on the lipid bilayer sensor.
"We’ve accomplished many of our goals," Bob says. "We’ve been able to build rugged lipid bilayers that last as long as three weeks. We’ve figured out how to introduce ion channels into the lipid bilayers. We’ve proven you can make ion channels selective to certain ions in the solution. Now we have to attach antibodies to the ion channels to show we can detect different biological agents. The antibody work may be our most difficult yet."