In order to advance in our understanding of biological systems we need better tools for measuring what goes on in cells and between cells. Tools that let us watch more things at once at a smaller scale, for longer periods of time and with greater sensitivity can greatly speed up the rate at which the functioning of biological systems can be puzzled out. Quantum dots can do all those things as a number of recent reports have shown.
A team at Rockefeller University and the US Naval Research Laboratory have developed a way to use quantum dots to label different kinds of proteins in living cells to fluoresce at different colors so that the internal components of cells can be tracked and imaged for long periods of time.
Quantum dots are nano-sized crystals that exhibit all the colors of the rainbow due to their unique semiconductor qualities. These exquisitely small, human-made beacons have the power to shine their fluorescent light for months, even years. But in the near-decade since they were first readily produced, quantum dots have excluded themselves from the useful purview of biology. Now, for the first time, this flexible tool has been refined, and delivered to the hands of biologists.
Quantum dots are about to usher in a new plateau of comparative embryology, as well as limitless applications in all other areas of biology.
Two laboratories at The Rockefeller University -- the Laboratory of Condensed Matter Physics, headed by Albert Libchaber, Ph.D., and the Laboratory of Molecular Vertebrate Embryology, headed by Ali Brivanlou, Ph.D. -- teamed up to produce the first quantum dots applied to a living organism, a frog embryo. The results include spectacular three-color visualization of a four-cell embryo.
The scientists' results appear in the Nov. 29 issue of Science.
"We always knew this physics/biology collaboration would bear fruit," says co-author Brivanlou, "we just never knew how sweet it would be. Quantum dots in vivo are the most exciting, and beautiful, scientific images I have ever seen."
To exploit quantum dots' unique potential, the Rockefeller scientists needed to make a crucial modification to existing quantum dot technology. Without it, frog embryos and other living organisms would be fallow ground for the physics-based probes.
"Quite simply, we cannot do this kind of cell labeling with organic fluorophores," says Brivanlou. Organic fluorophores (synthetic molecules such as Oregon Green and Texas Red) don't have the longevity of quantum dots. What's more, organic fluorophores and fluorescent proteins (such as green fluorescent protein, a jellyfish protein, and luciferase, a firefly protein) represent a small number of colors, subject to highly specific conditions for effectiveness. Quantum dots can be made in dozens of colors just by slightly varying their size. The application potential in embryology alone is monumental.
Hydrophobic, but not claustrophobic
Benoit Dubertret, Ph.D., a postdoctoral fellow working with Libchaber, toiled for two years with quantum dots' biggest problem: their hydrophobic (water-fearing) outer shell. This condition, a by-product of quantum dots' synthesis, makes them repellent to the watery environment of a cell, or virtually any other biological context.
The ability to do track cells as they differentiate has enormous value for the development of stem cell therapies and the growth of replacement organs.
These scientists have developed the ability to have the cells take up the quantum dots using endocytosis so that injection into a cell is no longer necessary. They have also developed a way to link quantum dots to antibodies that have affinity to specific proteins.
The unique physical properties of quantum dots overcome these obstacles. Simply by altering their size, scientists can manufacture them to produce light in any color of the rainbow, and, additionally, only one wavelength of light is required to illuminate all of the different-colored dots. Thus, spectral overlap no longer limits the number of colors that can be used at once in an experiment. In addition, quantum dots do not stop glowing even after being visualized for very long periods of time: compared to most known fluorescent dyes, they shine for an average of 1,000 times longer.
But while quantum dots solve these problems, they have limitations of their own - the biggest one being their water-fearing or "hydrophobic" nature. For quantum dots to mix with the watery contents of a cell, they have to possess a water-loving, or "hydrophilic" coat. Three years ago, Simon and Jaiswal's colleagues at the U.S. Naval Research Laboratory made their dots biocompatible by enveloping them in a layer of the negatively charged dihydroxylipoic acid (DHLA).
In the same study, the researchers overcame a second major obstacle of making quantum dots biologically useful - building protein-specific dots. By linking antibodies specific for an experimental protein to the DHLA-capped dots, they were able to demonstrate protein-specificity in a test tube.
In the present study, the Rockefeller scientists in collaboration with their U.S. Naval Research Laboratory colleagues have again synthesized protein-specific quantum dots, but this time they have shown their efficacy in living cells - a first for this budding technology. To do this, the researchers employed two different methods of synthesizing the quantum dots, both of which involved linking the negatively charged DHLA-capped dots to positively charged molecules - either avidin or protein G bioengineered to bear a positively charged tail. Because avidin and protein G can be made to readily bind antibodies, the researchers could then attach the dots to their protein-specific antibody of choice.
The critical test was to determine specificity: can quantum dots achieve the same exquisite selectivity that occurs when a protein is synthesized fused to GFP? To answer this question, Simon and colleagues engineered a population of cells growing together in a dish to randomly produce different levels of a membrane protein fused to GFP. When these cells were incubated with quantum dots conjugated to an antibody specific for that membrane protein, the pattern of GFP fluorescence matched the fluorescence of the quantum dots. However, the fluorescence of quantum dots lasted immeasurably longer, and the proteins could now be imaged in a rainbow of colors.
"Researchers should now be able to rapidly create an assortment of quantum dots that specifically bind to several proteins of interest," says Jaiswal.
Uncharted cellular terrainProteins aren't the only subjects the researchers successfully lit up with quantum dots: cells too were labeled and observed in their normal setting for very long periods of time. In the Nature Biotechnology paper, the researchers monitored human tissue culture cells tagged with quantum dots over two weeks with no adverse effects on cells. They also continuously observed slime mold cells labeled with quantum dots through 14 hours of growth and development without detecting any damage. This type of cell-tracking approach would allow researchers to study cell fate either outside the body in culture, or in whole developing organisms.
Quantum Dot Corporation researchers use quantum dots to detect cancer cells.
Hayward, CA, December 2, 2002 - Quantum Dot Corporation (QDC), the leader in Qdot(tm) biotechnology applications and products, announced today the publication of a seminal scientific paper in the prestigious journal Nature Biotechnology. The paper, entitled "Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots", was published in the on-line version of Nature Biotechnology, following collaborative work performed by scientists at Genentech and QDC. The print version will be published in January 2003."The promise of Qdot conjugates to revolutionize biological detection has now become a reality. Our work with Genentech is the first practical application of the Qdot technology in an important biological system - specific detection of breast cancer markers. These results demonstrate the dramatic sensitivity and stability benefits enabled using Qdot detection," said Xingyong Wu, Ph.D., senior staff scientist at QDC, and the lead author of the paper. "We have also demonstrated cancer marker detection in live cancer cells, an extremely difficult task using conventional methods," continued Dr. Wu.
Small Times has an article that provides an overview of some of these recent results with quantum dots.
A third team of researchers reported their solution to the biocompatibility problem in Science. They sheathed the dots in phospholipid membranes and hooked them to DNA to produce clear images in growing embryos, where the nanocrystals appeared stable and nontoxic.
"These three papers combined indicate that bioconjugate nanocrystals will have major applications in biology and medicine," said Shuming Nie, director of nanotechnology at Emory University's Winship Cancer Institute.
Emory University biomedical engineer Shuming Nie argues that nanotechnology will provide benefits for biomedical applications many years before nanotech becomes beneficial in electronics applications.
Biomedical engineer Shuming Nie is testing the use of nanoparticles called quantum dots to improve clinical diagnostic tests for the early detection of cancer. The tiny particles glow and act as markers on cells and genes, potentially giving scientists the ability to rapidly analyze biopsy tissue from cancer patients so that doctors can provide the most effective therapy available.
Nie, a chemist by training, is an associate professor in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University and director of cancer nanotechnology at Emory's Winship Cancer Institute.
His research focuses on the field of nanotechnolgy, in which scientists build devices and materials one atom or molecule at a time, creating structures that take on new properties by virtue of their miniature size. The basic building block of nanotechnology is a nanoparticle, and a nanometer is one-billionth of a meter, or about 100,000 times smaller than the width of a human hair.
Nanoparticles take on special properties because of their small size. For example, if you break a piece of candy into two pieces, each piece will still be sweet, but if you continue to break the candy until you reach the nanometer scale, the smaller pieces will taste completely different and have different properties.
Until recently, nanotechnology was primarily based in electronics, manufacturing, supercomputers and data storage. However, Nie predicted several years ago in a paper published in Science that the first major breakthroughs in the field would be in biomedical applications, such as early disease detection, imaging and drug delivery.
"Electronics may be the field most likely to derive the greatest economic benefit from nanotechnology," Nie said. "However, much of the benefit is unlikely to occur for another 10 to 20 years, whereas the biomedical applications of nanotechnology are very close to being realized."
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