SCIENCE & TECHNOLOGY beads is a partitioning process," Nie says. "For the quantum dot to go into the polystyrene bead, we have to make the quantum dot's surface match the hydrophobicity of the interior of the polymer bead."
BAR CODING' BIOMOLECULES Quantum-dot-tagged polymeric microbeads provide multiplexed optical coding C E L I A M. H E N R Y , C & E N W A S H I N G T O N
M
ICROBEADS EMBEDDED WITH
precisely controlled ratios of luminescent semiconductor nanocrystals—also known as quantum dots—can be used as "bar codes" to identify molecules in bioassays. The optical codes are based on different sizes (and thus colors) and intensities of quantum dots. The codes can be used for high-throughput analysis of biological molecules, say chemists at Indiana University, Bloomington [Nat. Biotechnol., 19,631(2001)}. The nanocrystals offer several advantages over organic dyes, according to the team—associate chemistry professor Shuming Nie, postdocs Mingyong Han and Jack Z. Su, and graduate student Xiaohu Gao. A single excitation wavelength can be used for all of the quantum dots, and their emission wavelengths vary according to their size. In addition, the emission profile of the quantum dots is
perfectly symmetric, which increases the capacity for multiplexing—that is, identifying many wavelengths at once. For a single color incorporated in microbeads, 10 intensity levels can be clearly distinguished, Nie says. As more colors are added to the beads, differentiating intensity levels becomes increasingly difficult. As the number of colors is increased, the number of intensity levels should be decreased for optimum performance, Nie recommends. "It's much easier to distinguish the frequency or color than to distinguish the absolute intensity" Nie says. 'The intensity can be affected by many things, including instrument response and alignment. It's better to use more colors" than more intensities. Nie and his coworkers embed hydrophobic ZnS-capped CdSe quantum dots into polymeric beads. They previously worked with water-soluble quantum dots. "Putting the quantum dots into the Fluorescence intensity Optical code 1:1:1 No
iiiiiiiiiiiii Probe #1
Single-bead spectroscopy
analyte
A
A
A
1:1:1 Target #2
©
IIIIIIIIIIIIC
Single-bead spectroscopy^
Probe #2
Analyte
r
AAA
^*
1:2:1
Target #3 p
Analyte
u n n n x m c Single-bead spectroscopy E Probe #3 — a ^ 2:1:1
Target #4 J£> iIIIIII11iiQZT Single-bead spectroscopy^ Probe #4 *
A
TAAA Wavelength (nm)
MULTIPLEXING DNA hybridization assays can be carried out using beads tagged with quantum dots. Probe oligonucleotides are conjugated to the tagged beads. Target sequences must be tagged as well with either a fluorescent dye or a quantum dot. The probe sequences are identified by the absolute intensity and the ratios of the quantum-dot fluorescence emission. 24
C & E N / JULY 16, 2001
FOR THE CODES to be easily identifiable, the quantum dots must not aggregate, which could lead to spectral broadening, wavelength shifting, and energy transfer. Under such circumstances, the intensity is no longer a linear function of the number of quantum dots. However, energy transfer has not proved to be a problem. Nie believes that the porous structure of the beads separates the quantum dots. iCWe synthesize these beads with a large number of very small pores. The quantum dots move into these pores. Then we use a solvent to shrink the pores and trap the quantum dots," Nie says. To reduce the chances of aggregation even further, Nie and his coworkers load the quantum dots in descending order of size, starting with the largest dots—the ones that emit red light—first. Another possibility, Nie says, is simply that there are many more pores than quantum dots in the beads, leaving the majority of the pores empty The chemists demonstrate three-color optical bar codes with a D N A oligonucleotide assay. Probe oligonucleotide sequences are conjugated to the quantumdot-tagged microbeads. Fluorescently labeled target D N A sequences are then allowed to hybridize to the probes. The beads are analyzed by single-bead spectroscopy in which signals for both the code and the analyte are detected. "In a real assay, the encoded beads are mixed and are incubated with the same sample. Optical readout is achieved by single-bead spectroscopy or single-bead imaging," Nie says. "It's like a bar code reader. When you check out items in a store, they are scanned one at a time." In an assay, this could be done in a fashion similar to flow cytometry Another possibility, Nie says, would be to use magnetic materials to force the beads into a monolayer in a microliter plate. The monolayer could be read with a laser beam similarly to compact discs. Spectral overlap is a potential hurdle to increasing the number of available optical codes. "If you put too many colors, they will overlap. If you have that overlap, you will need some kind of deconvolution software or algorithms. \ b u will need to do curve fitting to resolve those peaks. These technical hurdles can be overcome, especially by using software to recognize the codes," Nie says. HTTP://PUBS.AC5.ORG/CEN
Quantum Dot Corp. in Hayassay signal is seen only on the ward, Calif., is doing similar work. beads carrying the anticytokine Marcel Bruchez, a scientist at antibodies. Quantum Dot, described some of "One of the keys that distinthe company's work at the Amerguishes this technique from flow ican Chemical Society's ProSpeccytometry is that we don't need to tives Conference on Biological use thousands to tens of thousands Applications of Nanotechnology, of clear beads to get our signal," held last month in Berkeley, Calif. Bruchez said. "We can use hunThe company has developed a dreds of beads, and as long as we new chemistry for biological get agood statistical sampling of all applications using quantum dots. of the beads in the sample, we can Researchers adsorb a modified calculate roughly how many beads polyacrylic acid onto the surface we have." He pointed out that this of the quantum dot. "We wind up BRIGHT LIGHTS Different colors of quantum dots improves the sensitivity with a structure that is soluble in can be easily distinguished. This fluorescence Nie, meanwhile, is working to water, but neither of the original micrograph shows a mixture of CdSe/ZnS QD-tagged extend the spectral range of quancomponents is particularly soluble beads emitting single-color signals at 484p 508, 547, tum dots. "We think that near-IR in water," Bruchez said. 575, and 611 nm. quantum dots might be even better. They emit at much longer Quantum Dot is also incorpowavelengths. The background essentially Bruchez described a protein-profiling rating the nanocrystals into latex beads disappears because very few compounds assay in which five anticytokine antibodfor multiplexing and creating optical fluoresce in the near infrared," Nie says. ies are conjugated to beads with different codes. They've distinguished three intenHe and his coworkers have made quantum codes. Other antibodies, not related to sity levels in single-color beads, Bruchez dots that emit in the near-IR up to 850 cytokines, are conjugated to other coded said. Currently, they use eight colors to nm. That work, Nie says, has been subbeads. The beads are mixed with a sample make more than 100 codes. The beads mitted to theJournal of theAmerican Chemcontaining the five cytokines. The codes with four colors per bead are combined, ical Society. • and the analyte signals are then read; the but only a single intensity level is used.
Nano Letters invites original reports of fundamental research in all branches of the theory and practice of nanoscience and nanotechnology. CALL FOR PAPERS ANO LETTERS provides rapid disclosure of the key elements of a study, publishing preliminary, experimental, and theoretical results on the physical, chemical, and biological phenomena, processes and applications of structures within the nanoscale size range.
AJSUMAlimifB lOIAlOKIMa UIOHCMOIOET
=NAN0
ER S
This is the second letters journal launched by ACS, following the 1999 release of Organic Letters, and charged with the same mission: To rapidly communicate preliminary significant research results. EDITOR: A. Paul Alhrisatos, Professor of Chemistry and Materials Science and Mineral Engineering, University of California, Berkeley Submit your Paper with Multimedia Images Nano Letters supports a variety of file formats that allow authors to submit multimedia attachments to enhance the reader's understanding of the published manuscript. Readers of the Web Edition of Nano Letters will be able to view these multimedia items with an appropriate viewer from within the text of the manuscript.
Sample issue: http://pubs.acs.org/NanoLett Important Notes for Authors • NanoLetters publishes color images at the discretion of the Editor and at no cost to the author • There are no page charges associated with NanoLetters • Fifty free electronic reprints are available to every author
HTTP://PUBS.ACS.ORG/CEN
SUBMIT YOUR MANUSCRIPT ELECTRONICALLY! Nano Letters offers authors the flexibility of electronic manuscript submission via the Web. Complete instructions are at the journal website, http://pubs.acs.org/NanoLett
ACS PUBLICATIONS HIGH
QUALITY. HIGH IMPACT. http://pubs.acs.org
C&EN
/ JULY
16. 2001
25
