Multiplexed SNP genotyping using the Qbead system: a quantum dot-encoded microsphere-based assay.

We have developed a new method using the Qbead system for high-throughput genotyping of single nucleotide polymorphisms (SNPs). The Qbead system employs fluorescent Qdot semiconductor nanocrystals, also known as quantum dots, to encode microspheres that subsequently can be used as a platform for multiplexed assays. By combining mixtures of quantum dots with distinct emission wavelengths and intensities, unique spectral 'barcodes' are created that enable the high levels of multiplexing required for complex genetic analyses. Here, we applied the Qbead system to SNP genotyping by encoding microspheres conjugated to allele-specific oligonucleotides. After hybridization of oligonucleotides to amplicons produced by multiplexed PCR of genomic DNA, individual microspheres are analyzed by flow cytometry and each SNP is distinguished by its unique spectral barcode. Using 10 model SNPs, we validated the Qbead system as an accurate and reliable technique for multiplexed SNP genotyping. By modifying the types of probes conjugated to microspheres, the Qbead system can easily be adapted to other assay chemistries for SNP genotyping as well as to other applications such as analysis of gene expression and protein-protein interactions. With its capability for high-throughput automation, the Qbead system has the potential to be a robust and cost-effective platform for a number of applications.

Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots.

Semiconductor quantum dots (QDs) are among the most promising emerging fluorescent labels for cellular imaging. However, it is unclear whether QDs, which are nanoparticles rather than small molecules, can specifically and effectively label molecular targets at a subcellular level. Here we have used QDs linked to immunoglobulin G (IgG) and streptavidin to label the breast cancer marker Her2 on the surface of fixed and live cancer cells, to stain actin and microtubule fibers in the cytoplasm, and to detect nuclear antigens inside the nucleus. All labeling signals are specific for the intended targets and are brighter and considerably more photostable than comparable organic dyes. Using QDs with different emission spectra conjugated to IgG and streptavidin, we simultaneously detected two cellular targets with one excitation wavelength. The results indicate that QD-based probes can be very effective in cellular imaging and offer substantial advantages over organic dyes in multiplex target detection.

Excited-State Electronic Structure in Polypyridyl Complexes Containing Unsymmetrical Ligands.

Step-scan Fourier transform infrared absorption difference time-resolved (S(2)FTIR DeltaA TRS) and time-resolved resonance Raman (TR(3)) spectroscopies have been applied to a series of questions related to excited-state structure in the metal-to-ligand charge transfer (MLCT) excited states of [Ru(bpy)(2)(4,4'-(CO(2)Et)(2)bpy)](2+), [Ru(bpy)(2)(4-CO(2)Et-4'-CH(3)bpy)](2+), [Ru(bpy)(4,4'-(CO(2)Et)(2)bpy)(2)](2+), [Ru(4,4'-(CO(2)Et)(2)bpy)(3)](2+), [Ru(bpy)(2)(4,4'-(CONEt(2))(2)bpy)](2+), [Ru(bpy)(2)(4-CONEt(2)-4'-CH(3)bpy)](2+), and [Ru(4-CONEt(2)-4'-CH(3)bpy)(3)](2+) (bpy is 2,2'-bipyridine). These complexes contain bpy ligands which are either symmetrically or unsymmetrically derivatized with electron-withdrawing ester or amide substituents. Analysis of the vibrational data, largely based on the magnitudes of the nu(CO) shifts of the amide and ester substituents (Deltanu(CO)), reveals that the ester- or amide-derivatized ligands are the ultimate acceptors and that the excited electron is localized on one acceptor ligand on the nanosecond time scale. In the unsymmetrically substituted acceptor ligands, the excited electron is largely polarized toward the ester- or amide-derivatized pyridine rings. In the MLCT excited states of [Ru(bpy)(2)(4,4'-(CO(2)Et)(2)bpy)](2+) and [Ru(bpy)(2)(4,4'-(CONEt(2))(2)bpy)](2+), Deltanu(CO) is only 60-70% of that observed upon complete ligand reduction due to a strong polarization interaction in the excited state between the dpi(5) Ru(III) core and the excited electron.

A New Electron-Transfer Donor for Photoinduced Electron Transfer in Polypyridyl Molecular Assemblies.

A synthetic procedure has been devised for the preparation of the reductive quencher ligand 4-methyl-4'-(N-methyl-p-tolylaminomethyl)-2,2'-bipyridine (dmb-tol), which contains toluidine covalently bound to 2,2'-bipyridine. When bound to Re(I) in [Re(I)(dmb-tol)(CO)(3)Cl], laser flash Re(I) --> dmb metal-to-ligand charge-transfer (MLCT) excitation at 355 nm in CH(3)CN at 298 +/- 2 K is followed by efficient, rapid (<5 ns) appearance of a transient with an absorption feature at 470 nm. The transient spectrum is consistent with formation of the redox-separated state, [Re(I)(dmb(-)-tol(+))(CO)(3)Cl], which returns to the ground state by back electron transfer with k(ET) = (1.05 +/- 0.01) x 10(7) s(-)(1) (tau = 95 +/- 1 ns) at 298 +/- 2 K. Rapid, efficient quenching is also observed in the Ru(II) complex [Ru(4,4'-(C(O)NEt(2))(2)bpy)(2)(dmb-tol)](2+). Based on transient absorption measurements, a rapid equilibrium appears to exist between the initial metal-to-ligand charge-transfer excited state and the redox-separated state, which lies at higher energy. Decay to the ground state is dominated by back electron transfer within the redox-separated state which occurs with k > 4 x 10(8) s(-)(1) at 298 +/- 2 K.

Mid-Infrared Spectrum of [Ru(phen)(3)](2+).

Time-resolved infrared spectra in the fingerprint region (1300-1700 cm(-)(1)) are reported for the metal-to-ligand charge-transfer (MLCT) excited state(s) of [Ru(phen)(3)](2+) and [Os(phen)(DAS)(2)](2+) (phen is 1,10-phenanthroline; DAS is 1,2-bis(diphenylarsino)ethane) in acetonitrile-d(3) at 298 K. The spectra are assigned by comparison to electrochemically generated [Ru(III)(phen)(3)](3+) and [Ru(II)(phen(*)(-)())(phen)(2)](+). The data provide clear evidence for the localized description [Ru(III)(phen(*)(-)())(phen)(2)](2+) on the approximately 100 ns time scale. They also give insight into electronic distribution in the excited state, aid in the interpretation of the time-resolved resonance Raman spectrum of [Ru(phen)(3)](2+), and suggest why measuring ground- and excited-state resonance Raman spectra of phen complexes is difficult.

Synthesis, Characterization, and Photochemical/Photophysical Properties of Ruthenium(II) Complexes with Hexadentate Bipyridine and Phenanthroline Ligands.

Three hexadentate, podand-type, polypyridyl ligands, (5-bpy-2C)(3)Bz, (4-bpy-2C-Ph)(3)Et, and (4-phen-2C-Ph)(3)Et, and their Ru(II) and Fe(II) complexes have been prepared. Reaction of these ligands with Fe(II) produces only the monometallic hemicage species, while monometallic, bimetallic, and polymetallic Ru(II) complexes are formed. These species are separable by column chromatography, and NMR and ESI mass spectrometry demonstrate that with each ligand the first band to elute corresponds to the monometallic species, [RuL](2+). The ESI mass spectra show peaks for [RuL](2+) and [RuL](PF(6))(+) with expected m/z values and isotope peak spacings. (1)H NMR spectroscopy shows that [Ru(5-bpy-2C)(3)Bz](2+) is trigonally symmetric and contains a rigid methylene bridge between the capping group and the bipyridines. The excited-state lifetimes and emission quantum yields for the hemicage complexes, [Ru(5-bpy-2C)(3)Bz](2+), [Ru(4-bpy-2C-Ph)(3)Et](2+), and [Ru(4-phen-2C-Ph)(3)Et](2+), are significantly enhanced (tau = 2800, 1470, and 3860 ns, and Phi(em) = 0.271, 0.104, 0.202, respectively) relative to the model compounds and to the polymetallic complexes with the same ligand. An Arrhenius fit of temperature-dependent lifetime data for [Ru(5-bpy-2C)(3)Bz](2+) indicates a high activation energy for crossover to the dd state (DeltaE = 4960 cm(-)(1)) as well as the existence of an additional pathway for deactivation via a "4th MLCT" state. Only after extensive photolysis of [Ru(5-bpy-2C)(3)Bz](2+) is any decrease in emission intensity observed; this is accompanied by the formation of a bimetallic photoproduct, [Ru(2)L(2)](4+), with a quantum yield of 7.4 x 10(-)(6). Quenching studies with a variety of quenchers show that the useful excited-state redox and energy-transfer properties characteristic of Ru(II) polypyridyls are retained, but with improved photoinertness and photophysical properties arising from the rigidity of the hemicage complex.

Effect of Delocalization and Rigidity in the Acceptor Ligand on MLCT Excited-State Decay.

In its most simple form, the energy gap law for excited-state nonradiative decay predicts a linear dependence of ln k(nr) on the ground- to excited-state energy gap, where k(nr) is the rate constant for nonradiative decay. At this level of approximation, the energy gap law has been successfully applied to nonradiative decay in a wide array of MLCT excited states of polypyridyl complexes of Re(I), Ru(II), and Os(II). This relationship also predicts a dependence of k(nr) on the structural characteristics of the acceptor ligand. We report here a brief survey of the literature which suggests that such effects exist and have their origin in the extent of delocalization of the excited electron in the ligand pi framework and on acceptor ligand rigidity.