This article provides a comprehensive overview of the progress made in the past eight years in implementing the use of luminescent semiconductor quantum dots (QDs) in biological applications. We believe that researchers are turning to this article to learn what has been accomplished, to see if some development would be applicable to their work, and also for a critical assessment of the field in general.

“...the group was able to characterize how QDs functioned as resonance energy-transfer donors and then to incorporate them into several different biosensing configurations.”

QD-bioconjugates represent one of the more mature nanotechnologies where materials, chemistry, and physics interface with biology with tangible new results. As such, there is tremendous interest in these materials from a variety of different disciplines. These can range from related biological fields such as cellular research, drug discovery, and clinical diagnostics, to emerging areas of nanotechnology such as molecular electronics and alternate energy generation.

As QD usage increases, physical chemists, biologists, and many others are interested in finding out whether utilizing their unique properties can further their research or improve their assay capabilities. For example, those working on developing diagnostics methods and assays are interested in using them for creating improved nanoscale sensors, while electric engineers may be interested in bioinspired electronics.

This is a review article with a focus on assessing the progress made in developing biological applications of QD nanoprobes. It describes what the materials are, how they are made, their unique features, why biologists in particular would be interested in using them, and what can be achieved though employing them. It also describes some of the more prominent biological demonstrations to date.

More importantly the review provides a critical overview of two important and related areas:

1: We examine the available methods for creating QD-bioconjugates (QD-protein/DNA probes) along with a critical discussion of the advantages and disadvantages of each of these methods.

2: We examine many of the remaining issues that need to be addressed in order for full utilization of these materials in biological applications. We further explain how QDs are not meant to replace traditional fluorophores, but rather to complement and augment them in specialized areas. We also discuss some areas that will see a lot of progress in the near future, for example, "multiplexing" or the simultaneous assaying of multiple biological targets.

The most common method of labeling, visualizing, and quantifying biological molecules and processes in solution and inside cells is through the use of dye-based fluorescent labeling. Dyes have advanced the field and allowed a multitude of phenomena to be discovered and understood.

They do, however, have certain properties that can limit their usage. For example they can be prone to bleaching or chemical and biological degradation. QDs have many unique optical and spectroscopic properties and are much more resistant to chemical and photodegradation than commonly used dyes.

Relatively larger than molecular dyes, their nanometer size means that they have a much larger absorption cross-section than commonly used organic dyes (they can absorb/emit more light) and accessible surface area for subsequent attachment of molecules (they can attach more biomolecules to their surface).

When these unique properties are used correctly, QDs can be far superior to the standard chemical dyes currently in biological use, especially from an optical standpoint. For instance, you can use one light source to illuminate many different colors of QDs very effectively; something that is very hard to achieve with conventional dyes.

QD nanoprobes (nanometer size semiconductor crystals interfaced with biological molecules) are a good representative of what nanotechnology has to offer scientifically. However, integrating these nanosized materials into biological applications is a continuous learning process.

Thus researchers from many fields are interested in how people are using these materials in biology, what approaches they are taking to make them work for their applications, and perhaps, most importantly, what are the key scientific issues to address and understand when working with QDs in biology.

Igor Medintz’s background graduate work is in classical molecular biology using yeast as a model organism. Igor then did post-doctoral research with Professor Richard Mathies in the Chemistry Department at the University of California, Berkeley. This exposed him to the world of microfabricated "lab-on-a-chip" devices and integrating biological assays into them.

A major portion of this research was focused on new fluorophores and resonance energy transfer to help simplify analysis on these devices. He had also worked in a clinical diagnostics laboratory for several years. In early 2002, Igor had an opportunity to do research at the U.S. Naval Research Laboratory (NRL) under a National Research Council Fellowship.

This opportunity exposed him to both QDs and the development of biosensing technology and he immediately realized that there was a tremendous potential for fundamental synergistic research between the biological and materials fields.

Hedi Mattoussi initiated the project on QDs and their use in biology shortly after moving to NRL. He was able to combine his background in materials and physics (including QD synthesis and characterization, understanding of their fundamental physical properties, and polymer physics) with a strong desire to cross the "disciplinary divide" into biological applications.

In addition, NRL has a strong commitment to supporting new optical materials and developing biosensing technologies. This allowed us to eventually form what is currently a small but committed interdisciplinary research group, to move into several new areas, and to initiate some important collaborations. In particular, the group was able to characterize how QDs functioned as resonance energy-transfer donors and then to incorporate them into several different biosensing configurations.

Since biologically compatible QDs are a relatively new class of materials, learning to use these "first generation" materials has presented the most challenges. From the materials perspective, some of the issues that had to be dealt with included developing methods for attaching biomolecules (proteins) to QDs, controlling the number of proteins attached to each QD, and controlling their orientation as well as relative distance to the QD surface.

From the biological perspective, proteins had to be engineered to interact with the QDs and assay conditions had to be developed that were compatible for both. Learning to exploit the larger size of the QDs relative to fluorophores as a benefit and not a liability was another challenge. We accomplish this by utilizing the QD as a central nanoscaffold. We then attach multiple biological entities such as proteins around the QD, which can increase sensitivity and signal when used correctly.

Many of the considerations when working with these materials are the same as those that apply to the burgeoning field of nanotechnology and new nanoscale materials. The scientific community has still not devised a systematic nomenclature for classifying and naming all the different nanoparticles and new materials. Additionally, there are still many things to be learned about their long-term toxicity.