NEWS AND VIEWS

http://biotech.nature.com • JULY 2001 • VOLUME 19 • nature biotechnology

Three years ago, nanocrystals (or quantumdots, QDs) were first demonstrated capableof labeling specific components of cells andtracing proteins within cells1,2. Since then,several groups have been attempting tooptimize fluorescent nanocrystals into alabeling system that can be practicallyapplied to a variety of biological applica-tions. In this issue, Han et al.3 report a fur-ther extension of the biological utility ofsemiconductor quantum dots to includeassays based on microbeads embedded withdifferent sizes (and thus different fluores-cent colors) of dots in controlled ratios,essentially creating a QD bar code(http://www.qdots.com/noflash/technolo-gy/beadtech.html). Because of the uniquespectral properties of QDs, this technologyhas potentially tremendous multiplexingcapability and might therefore find com-mercial application in genomics, pro-teomics, and high-throughput screening.

Fluorescent QDs consist of a core of acadmium selenide (CdSe) nanocrystalranging in diameter from 18 Å (∼ 200atoms) to 70 Å (∼ 10,000 atoms). CdSenanocrystals have a low fluorescent quan-tum yield because the optically excited car-riers (a positive and negative charge) gettrapped at the poorly passivated surface ofthe nanocrystal (surface atoms bonded toonly three other atoms rather than four).However, when the core is wrapped in ashell of several monolayers of zinc sulfide(ZnS), the core/shell crystals have a fluo-rescent quantum yield of 50%. The extinc-tion coefficient of the nanocrystals is sever-al times larger than an organic dye mole-cule, making the nanocrystals incrediblybright. The size of the core controls theabsorption and emission wavelengththrough a quantum mechanical confine-ment energy of the optical excitation—hence the nickname “quantum dot”. Thesmaller the nanocrystal, the greater theconfinement energy, and the higher theenergy of light it absorbs.

An 18 Å core absorbs and emits in the

blue spectrum, whereasa 70 Å core emits in thered, and the absorptionand emission wave-length are size-tunablein between (see Fig. 1).The absorption spec-trum, however, is con-tinuous above the firstexcitation peak. This isthe first advantage thatQD microbeads haveover their organic fluo-rophore counterparts:a single wavelength, say400 nm, will excite allof the QDs, whereasmultiple wavelengthswould be necessary toexcite organic dye mol-ecule–doped beads.

Second, the size dis-tribution of the coredictates the width ofthe emission spectrum.Good preparationslead to a size distribu-tion of ±2 Å and a fullwidth at half-height of30 nm in the emissionspectrum. This is com-parable to the best organic fluorophores.However, organic fluorophores have a log-normal line shape with long tails extendingto the red. This limits multiplexing andwould require interference or bandpass fil-ters to be used in the optical readout for-mat, limiting the dynamic range of intensi-ty levels. In contrast, the emission spec-trum of QDs is gaussian. This advantage ofthe core/shell nanocrystals is exploited byHan et al. in the microbeads: because oftheir symmetrical, gaussian emission lineshapes, fluorescent QDs have greater mul-tiplexing capacity than beads doped withorganic fluorophores.

Finally, the high brightness of the QDs hasadvantages. It means that the dynamic rangeof intensity levels may be improved overbeads doped with organic fluorophores.

In their paper, Han et al.1 first prepareand analyze polystyrene microspheres (1.2µm) doped with a single size CdSe/ZnSquantum dot in fixed amounts.

Fluorescence intensity analysis of the beadscleanly discriminated ten different intensi-ty levels and indicated a high level of beaduniformity and bead identification accura-cy. Next, the authors prepared beads withthree colors of quantum dots. A significantresult is that Forster resonant energy transfer from the small nanocrystals to thelarge nanocrystals does not occur. Thetriple-coded bead was then used to demon-strate a DNA hybridization assay. Theseresults indicate the fluorescent nanocrys-tal-embeded microspheres represent a newspectral coding technology that has poten-tially wide application to a variety of bio-logical assays.

So how many codes are possible exploit-ing different sizes of dots at differentdopant levels in the microbeads? Five sizesof CdSe–ZnS core/shell nanocrystals couldspan the visible spectrum with minimalspectral filtering required in the bead read-out format. Combined with six intensity

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Sandra J. Rosenthal is assistant professor,Department of Chemistry, VanderbiltUniversity, Nashville, TN 37235(sjr@femto.cas.vanderbilt.edu).

Figure 1. Absorption and emission spectra of a series of size-selectedCdSe nanocrystals. The CdSe core controls the spectral properties ofa CdSe–ZnS fluorescent quantum dot. As the size of the core increases, the absorption and emission wavelengthsshift to the red. However, as the absorption is continuous above thefirst excitation peak, one excitation source can be used to excite allsizes of core/shell quantum dots.

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Bar-coding biomolecules with fluorescent nanocrystalsMicrobeads embedded with quantum dots show promise as a multiplex coding technology for theanalysis of nucleic acids and proteins.

Sandra J. Rosenthal

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nature biotechnology • VOLUME 19 • JULY 2001 • http://biotech.nature.com

NEWS AND VIEWS

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levels, this would yield 10,000 codes.Additional sizes could be added withstricter filtering requirements, perhaps atthe cost of dynamic range of intensity levels.

The synthetic methodology forCdSe–ZnS quantum dots is now well estab-lished. The dots are robust and do not pho-todegrade. One major hurdle in commer-cialization is the difficulty of ensuringreproducible batch processing of theQD–tagged microbeads. Nevertheless, itappears that the availability of QD bar-coded beads is on the horizon3. The nexthurdle is to modify existing microbead for-mats to take advantage of this increase inmultiplexing capability.

This significant advance in optical bar-coding technology has potential to impactgenomics, proteomics, and other high-throughput screening assays. In comparisonto planar DNA chip technology, microbeadsoffer potentially improved hybridization,improved statistics, and increased flexibility.For example, typically 10–20 µl of unknown,labeled target DNA is deposited on a chip.Hybridization is limited by diffusional trans-port. The microbeads described by Han et al.could accelerate hybridization by allowingmore uniform mixing.

Second, DNA chip technology is typical-ly performed in duplicate or triplicate. Asthe QD microbeads can be assayed at20,000 per second, it would be reasonableto perform 50 or 100 replicates in a singleexperiment.

Finally, the DNA chips are a fixed assay(what’s on the chip is on the chip) whereasthe microbeads are a flexible assay, differ-ent beads can be “dialed in” to screen fordifferent genes based on the results of aprevious assay. In the realm of medicaldiagnoses, microbeads could be used toanalyze HIV titers, screen for drugs ofabuse, and test for allergies.

One significant obstacle remains beforeQDs have a wide impact in medical diag-nostics. CdSe–ZnS nanocrystals do notemit in the near infrared, so they cannot beused for analyses in blood. Nevertheless,new, robust nanocrystal methodologies areemerging rapidly and will make the con-cepts described by Han et al. applicable toeven a wider range of biological problems.

1. Bruchez, M. Jr., Moronne, M., Gin, P., Wiess, S. &Alivisatos, A.P. Science 281, 2031–2016 (1998).

2. Chan, W.C.W. & Nie, S., Science 281, 2016–2018(1998).

3. Han, M., Gao, X., Su, J.Z. & Nie, S. Nat. Biotechnol.19, 631–635 (2001).

Elegant and powerful technologies are usu-ally based on a simple concept. And it ishard to imagine a simpler concept thanmeasuring changes of ionic conductivitycaused by threading an RNA or DNA mole-cule through a membrane channel ornanopore1. Such measurements enabledirect, microsecond–time scale nucleicacid characterization without the need foramplification, chemical modification, sur-face adsorption, or the binding of probes

or intercalators2,3. Now two reports, one inthis issue by Howorka et al.4 and anotherpublished previously by Vecoutere et al.5,demonstrate the use of a nanopore tosequence a codon in a single molecule ofDNA or detect both single–base pair andsingle-nucleotide differences between molecules.

In both papers, the molecular amplifier6

used to detect single molecules is simplythe ionic current-carrying capacity of anelectrolyte-filled nanopore that spans aninsulating membrane (see Fig. 1). Whenthe nanopore is filled only with electrolyte(Fig. 1A), a voltage bias induces ions toflow through the nanopore. The currentflow is tiny (at the picoampere level), butreadily measured and recorded. When sin-gle- or double-stranded oligonucleotidesare drawn into the nanopore by the voltage

Nanopores with a spark for single-moleculedetectionProgress continues in exploiting ionic conductance of a membranechannel for single-molecule DNA sequence detection.

Hui Wang and Daniel Branton

bias, the oligonucleotides partially obstructthe nanopore and reduce its ion conductivi-ty (Fig. 1B, C). The reduction of ionic cur-rent is particularly striking when anoligonucleotide extends into, and blocks, aconstriction whose diameter is barely largeenough to allow translocation of theoligonucleotide’s narrowest dimension(Fig. 1D).

Both of the papers discussed here use α-hemolysin, a protein toxin produced by thebacterium Staphylococcus aureus, to form ananopore. The toxin self-assembles into alipid bilayer, as shown in Figure 1. By cova-lently tethering a DNA oligonucleotide nearthe entrance to the pore’s lumen, Howorkaand Bayley implement this molecularamplifier as a biosensor that can distinguishbetween polynucleotides whose sequenceperfectly complements that of the tetheredoligomer and those whose sequence imper-fectly complements, or does not comple-ment, the tethered oligomer. When a shortcomplementary polynucleotide is drawninto the lumen of the nanopore by a voltagebias, it is likely to form a DNA duplex withthe tethered oligomer, producing a charac-teristic current reduction that may last tensof milliseconds. On the other hand, apolynucleotide that does not complementthe tethered oligomer is rapidly drawn bythe voltage bias into the constriction of thetransmembrane region, producing a fleet-ing but marked current reduction that signals molecular translocation withoutduplex formation.

A series of experiments with polynu-cleotides each containing a differentsequence showed that, as expected, even asingle-base mismatch can influence the dis-tribution of duration times for duplex for-mation. When an individual oligonu-cleotide with an unknown codon sequenceis tethered to the nanopore and analyzed bythe application of a series of differentuntethered polynucleotides of knownsequence, the unknown codon sequence ofthe single tethered oligomer molecule canbe determined. Alternatively, if an array ofelectrically addressable nanopores with different known tethered oligonucleotidescould be implemented, such an array couldbe used in place of today’s surface-mountedmicroarrays to identify unknown sequencevariations in a solution of untetheredpolynucleotides.

Vercoutere et al. take a somewhat differ-ent approach and examine the duplexformed by the stems of molecules that takeon a hairpin conformation. They use hair-pins because the hairpin loops of thesemolecules adopt conformations that pre-vent entry into the nanopore lumen, thuskeeping the duplex stem portion of a hair-

Hui Wang is post doctoral research fellow andDaniel Branton is Research Professor ofBiology at the Department of Molecular andCellular Biology, The Biological Laboratories,16 Divinity Avenue, Harvard University,Cambridge, MA 02138(dbranton@harvard.edu).

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