Homogeneous time-resolved fluoroimmunoassay of bensulfuron-methyl by using terbium fluorescence energy transfer

A sensitive homogenous time-resolved fluoroimmunoassay (TR-FIA) method for bensulfuron-methyl (BSM) based on fluorescence resonance energy transfer (FRET) from a Tb(3+) fluorescent chelate with N,N,N('),N(')-[2,6-bis(3'-aminomethyl-1'-pyrazoly)-4-phenylpyridine] tetrakis(acetic acid) (BPTA-Tb(3+)) to organic dye, Cy3 or Cy3.5 has been developed. New method combined the use of BPTA-Tb(3+) labeled streptavidin, Cy3 or Cy3.5 labeled anti-BSM monoclonal antibody and biotinylated BSM-BSA conjugate (BSA is bovine serum albumin) for competitive-type immunoassay. After BPTA-Tb(3+) labeled streptavidin was reacted with a competitive immune reaction solution containing biotinylated BSM-BSA, BSM sample and Cy3 or Cy3.5 labeled anti-BSM monoclonal antibody, the sensitized and long-lived emission of Cy3 or Cy3.5 derived from FRET was measured, and thus the concentration of BSM in sample was calculated. The present method has the advantages of rapidity, simplicity and high sensitivity since the B/F (bound reagent/free reagent) separation steps and the solid-phase carrier are not necessary. The method gives the detection limit of 2.10 ngml(-1). The coefficient variations of the method are less than 1.5% and the recoveries are in the range of 95-105% for BSM water sample measurement.     

Design and characterization of two-dye and three-dye binary fluorescent probes for mRNA detection

We report the design, synthesis, and characterization of binary oligonucleotide probes for mRNA detection. The probes were designed to avoid common problems found in standard binary probes such as direct excitation of the acceptor fluorophore and overlap between the donor and acceptor emission spectra. Two different probes were constructed that contained an array of either two or three dyes and were characterized using steady-state fluorescence spectroscopy, time-resolved fluorescence spectroscopy, and fluorescence depolarization measurements. The three-dye binary probe (BP-3d) consists of a Fam fluorophore which acts as a donor, collecting light and transferring it as energy to Tamra, which subsequently transfers energy to Cy5 when the two probes are hybridized to mRNA. This design allows the use of 488 nm excitation, which avoids the direct excitation of Cy5 and at the same time provides a good fluorescence resonance energy transfer (FRET) efficiency. The two-dye binary probe system (BP-2d) was constructed with Alexa488 and Cy5 fluorophores. Although the overlap between the fluorescence of Alexa488 and the absorption of Cy5 is relatively low, FRET still occurs due to their close physical proximity when the probes are hybridized to mRNA. This framework also decreases the direct excitation of Cy5 and reduces the fluorescence overlap between the donor and the acceptor. Picosecond time-resolved spectroscopy showed a reduction in the fluorescence lifetime of donor fluorophores after the formation of the hybrid between the probes and target mRNA. Interestingly, BP-2d in the presence of mRNA shows a slow rise in the fluorescence decay of Cy5 due to a relatively slow FRET rate, which together with the reduction in the Alexa488 lifetime provides a way to improve the signal to background ratio using time-resolved fluorescence spectra (TRES). In addition, fluorescence depolarization measurements showed complete depolarization of the acceptor dyes (Cy5) for both BP-3d (due to sequential FRET steps) and BP-2d (due to the relatively low FRET rate) in the presence of the mRNA target.     

Fluorescence properties and photophysics of the sulfoindocyanine Cy3 linked covalently to DNA

The sulfoindocyanine Cy3 is one of the most commonly used fluorescent dyes in the investigation of the structure and dynamics of nucleic acids by means of fluorescence methods. In this work, we report the fluorescence and photophysical properties of Cy3 attached covalently to single-stranded and duplex DNA. Steady-state and time-resolved fluorescence techniques were used to determine fluorescence quantum yields, emission lifetimes, and fluorescence anisotropy decays. The existence of a transient photoisomer was investigated by means of transient absorption techniques. The fluorescence quantum yield of Cy3 is highest when attached to the 5' terminus of single-stranded DNA (Cy3-5' ssDNA), and decreases by a factor of 2.4 when the complementary strand is annealed to form duplex DNA (Cy3-5' dsDNA). Substantial differences were also observed between the 5'-modified strands and strands modified through an internal amino-modified deoxy uridine. The fluorescence decay of Cy3 became multiexponential upon conjugation to DNA. The longest lifetime was observed for Cy3-5' ssDNA, where about 50% of the decay is dominated by a 2.0-ns lifetime. This value is more than 10 times larger than the fluorescence lifetime of the free dye in solution. These observations are interpreted in terms of a model where the molecule undergoes a trans-cis isomerization reaction from the first excited state. We observed that the activation energy for photoisomerization depends strongly on the microenvironment in which the dye is located. The unusually high activation energy measured for Cy3-5' ssDNA is an indication of dye-ssDNA interactions. In fact, the time-resolved fluorescence anisotropy decay of this sample is dominated by a 2.5-ns rotational correlation time, which evidences the lack of rotational freedom of the dye around the linker that separates it from the terminal 5' phosphate. The remarkable variations in the photophysical properties of Cy3-DNA constructs demonstrate that caution should be used when Cy3 is used in studies employing DNA conjugates.     

Spectroscopic characterization of fluorescein- and tetramethylrhodamine-labeled oligonucleotides and their complexes with a DNA template

We measured absorption and emission spectra, fluorescence quantum yield, anisotropy, fluorescence resonance energy transfer (FRET), and melting temperature to characterize fluorescein- and tetramethylrhodamine (TMR)-labeled oligonucleotides in solution and when hybridized to a common DNA template. Upon hybridization to the template, both the absorption and emission spectra of TMR-labeled duplexes exhibited a shift with respect to those of labeled oligonucleotides, depending on the location of the TMR on the oligonucleotide. Measurements of quantum yield, anisotropy, and melting temperature indicated that TMR interacted with nucleotides within the duplexes in the order (T1>T5>T11, T16) that the oligonucleotide with TMR labeled at the 5' end (T1) is stronger than that labeled at position 5 from the 5' end (T5), which is also stronger than those labeled at the positions, 11 and 16, from the 5' end (T11, T16). In the case of the duplex formed between T1 and the template, fluorescence quenching was observed, which is attributed to the interaction between the dye molecule and guanosines located at the single-stranded portion of the template. A two-state model was suggested to describe the conformational states of TMR in the duplex. The melting temperatures of the four FRET complexes show the same pattern as those of TMR-labeled duplexes. We infer that the interactions between TMR and guanosine persist in the FRET complexes. This interaction may bring the donor and the acceptor molecules closely together, which could cause interaction between the two dye molecules shown in absorbance measurements of the FRET complexes.     

Subunit exchange of MjHsp16.5 studied by single-molecule imaging and fluorescence resonance energy transfer

MjHsp16.5 was separately labeled by fluorescent dye Cy3 and Cy5.5. The dissociation event of a single 24-mer MjHsp16.5 molecule was captured by single-molecule imaging (SMI). Temperature-regulated subunit exchange was revealed by the real-time fluorescence resonance energy transfer (FRET). The combination of single-molecular statistics and kinetic parameters from FRET experiments leads to the conclusion that below 75 degrees C the rate-determining step of the subunit exchange was the dissociation of the dye-labeled 24-mer in which the dimer was intact, whereas above 75 degrees C, smaller units emerged in the exchange and the rate-determining step had the character of a bimolecular reaction.     

Separation‐free single‐base extension assay with fluorescence resonance energy transfer for rapid and convenient determination of DNA methylation status at specific cytosine and guanine dinucleotide sites

A separation‐free single‐base extension (SBE) assay utilizing fluorescence resonance energy transfer (FRET) was developed for rapid and convenient interrogation of DNA methylation status at specific cytosine and guanine dinucleotide sites. In this assay, the SBE was performed in a tube using an allele‐specific oligonucleotide primer (i.e., extension primer) labeled with Cy3 as a FRET donor fluorophore at the 5′‐end, a nucleotide terminator (dideoxynucleotide triphosphate) labeled with Cy5 as a FRET acceptor, a PCR amplicon derived from bisulfite‐converted genomic DNA, and a DNA polymerase. A single base‐extended primer (i.e., SBE product) that was 5′‐Cy3‐ and 3′‐Cy5‐tagged was formed by incorporation of the Cy5‐labeled terminator into the 3′‐end of the extension primer, but only if the terminator added was complementary to the target nucleotide. The resulting SBE product brought the Cy3 donor and the Cy5 acceptor into close proximity. Illumination of the Cy3 donor resulted in successful FRET and excitation of the Cy5 acceptor, generating fluorescence emission from the acceptor. The capacity of the developed assay to discriminate as low as 10% methylation from a mixture of methylated and unmethylated DNA was demonstrated at multiple cytosine and guanine dinucleotide sites.     

A time-resolved luminescent competitive assay to detect L-selectin using aptamers as recognition elements

L-selectin is a protein with potential importance for numerous diseases and clinical disorders. In this paper, we present a new aptamer-based luminescent assay developed to detect L-selectin. The sensing system working principle is based on Förster Resonance Energy Transfer (FRET) from a donor terbium complex (TbC) to an acceptor cyanine dye (Cy5). In the present approach, the biotinylated aptamer is combined with Cy5-labelled streptavidin (Cy5-Strep) to yield an aptamer-based acceptor construct (Apta-Cy5-Strep), while L-selectin is conjugated using luminescent TbC. Upon aptamer binding to the TbC-labelled L-selectin (L-selectin-TbC), permanent donor-acceptor proximity is established which allows for radiationless energy transfer to occur. However, when unlabelled L-selectin is added, it competes with the L-selectin-TbC and the FRET signal decreases as the L-selectin concentration increases. FRET from the TbC to Cy5 was observed with time-gated time-resolved luminescence spectroscopy. A significant change in the corrected luminescence signal was observed in the dynamic range of 10–500 ng/mL L-selectin, the concentration range relevant for accelerated cognitive decline of Alzheimer's disease, with a limit of detection (LOD) equal to 10 ng/mL. The aptasensor-based assay is homogeneous and can be realized within one hour. Therefore, this method has the potential to become an alternative to tedious heterogeneous analytical methods, e.g. based on enzyme-linked immunosorbent assay (ELISA).     

Can luminescent quantum dots be efficient energy acceptors with organic dye donors?

We assessed the ability of luminescent quantum dots (QDs) to function as energy acceptors in fluorescence resonance energy transfer (FRET) assays, with organic dyes serving as donors. Either AlexaFluor 488 or Cy3 dye was attached to maltose binding protein (MBP) and used with various QD acceptors. Steady-state and time-resolved fluorescence measurements showed no apparent FRET from dye to QD. We attribute these observations to the dominance of a fast radiative decay rate of the donor excitation relative to a slow FRET decay rate. This is due to the long exciton lifetime of the acceptor compared to that of the dye, combined with substantial QD direct excitation.     

A new method for the detection of ATP using a quantum-dot-tagged aptamer

Fluorescence resonance energy transfer (FRET) between a quantum dot as donor and an organic fluorophore as acceptor has been widely used for detection of nucleic acids and proteins. In this paper, we developed a new method, characterized by 605-nm quantum dot (605QD) fluorescence intensity increase and corresponding Cy5 fluorescence intensity decrease, to detect adenosine triphosphate (ATP). The new method involved the use of three different oligonucleotides: 3′-biotin-modified DNA that binds to streptavidin-conjugated 605QD; 3′-Cy5-labelled DNA; and a capture DNA consisting of an ATP aptamer and a sequence which could hybridize with both 3′-biotin-modified DNA and 3′-Cy5-labelled DNA. In the absence of the target ATP, the capture DNA binds to 3′-biotin-modified DNA and 3′-Cy5-labelled DNA, bringing quantum dot and Cy5 into close proximity for greater FRET efficiency. When ATP is introduced, the release of the 3′-Cy5-labelled DNA from the hybridization complex took place, triggering 605QD fluorescence intensity increase and corresponding Cy5 fluorescence intensity decrease. Taken together, the virtue of FRET pair 605QD/Cy5 and the property of aptamer-specific conformation change caused by aptamer–ATP interaction, combined with the fluorescence intensity change of both 605QD and Cy5, provide prerequisites for simple and convenient ATP detection. Zhang Chen and Guang Li contributed equally to this work.     

New luminescent europium(III) chelates for DNA labeling

The new europium(III) chelate [2,2',2',2'-[[4'-(aminobiphenyl-4-yl)-2,2':6',2'-terpyridine- 6,6'-diyl]bis(methylenenitrilo)]tetrakis(acetato)] europium(III) (ATBTA-Eu3+) and its 4,6-dichloro-1,3,5-triazinyl and succinimidyl derivatives (DTBTA and NHS-ATBTA, respectively) were synthesized and characterized. Both labeling complexes DTBTA-Eu3+ and NHS-ATBTA-Eu3+ are luminescent. Especially DTBTA-Eu3+ is strongly luminescent, with a luminescence quantum yield of 9.1%, molar extinction coefficient of 3.1 x 10(4) cm(-1) M(-1) (335 nm), and luminescence lifetime of 1.02 ms. The excitation and emission maximum wavelengths of DTBTA-Eu3+ are 335 and 616 nm, respectively. The complex is very stable in aqueous buffers, with a conditional formation constant log K(DTBTA-Eu) of 25.0 at pH 8, and can be conjugated to DNA and proteins. The chelates are also highly resistant to thermal decomposition, photodegradation, and ozone oxidation. These properties prove that DTBTA-Eu3+ is suitable as a luminescence label in DNA assays.     

5' modification of duplex DNA with a ruthenium electron donor-acceptor pair using solid-phase DNA synthesis

Incorporation of metalated nucleosides into DNA through covalent modification is crucial to measurement of thermal electron-transfer rates and the dependence of these rates with structure, distance, and position. Here, we report the first synthesis of an electron donor-acceptor pair of 5' metallonucleosides and their subsequent incorporation into oligonucleotides using solid-phase DNA synthesis techniques. Large-scale syntheses of metal-containing oligonucleotides are achieved using 5' modified phosporamidites containing [Ru(acac)(2)(IMPy)](2+) (acac is acetylacetonato; IMPy is 2'-iminomethylpyridyl-2'-deoxyuridine) (3) and [Ru(bpy)(2)(IMPy)](2+) (bpy is 2,2'-bipyridine; IMPy is 2'-iminomethylpyridyl-2'-deoxyuridine) (4). Duplexes formed with the metal-containing oligonucleotides exhibit thermal stability comparable to the corresponding unmetalated duplexes (T(m) of modified duplex = 49 degrees C vs T(m) of unmodified duplex = 47 degrees C). Electrochemical (3, E(1/2) = -0.04 V vs NHE; 4, E(1/2) = 1.12 V vs NHE), absorption (3, lambda(max) = 568, 369 nm; 4, lambda(max) = 480 nm), and emission (4, lambda(max) = 720 nm, tau = 55 ns, Phi = 1.2 x 10(-)(4)) data for the ruthenium-modified nucleosides and oligonucleotides indicate that incorporation into an oligonucleotide does not perturb the electronic properties of the ruthenium complex or the DNA significantly. In addition, the absence of any change in the emission properties upon metalated duplex formation suggests that the [Ru(bpy)(2)(IMPy)](2+)[Ru(acac)(2)(IMPy)](2+) pair will provide a valuable probe for DNA-mediated electron-transfer studies.     

Intramolecular dimers: a new strategy to fluorescence quenching in dual-labeled oligonucleotide probes

Many genomics assays use profluorescent oligonucleotide probes that are covalently labeled at the 5' end with a fluorophore and at the 3' end with a quencher. It is generally accepted that quenching in such probes without a stem structure occurs through F?rster resonance energy transfer (FRET or FET) and that the fluorophore and quencher should be chosen to maximize their spectral overlap. We have studied two dual-labeled probes with two different fluorophores, the same sequence and quencher, and with no stem structure: 5'Cy3.5-beta-actin-3'BHQ1 and 5'FAM-beta-actin-3'BHQ1. Analysis of their absorption spectra, relative fluorescence quantum yields, and fluorescence lifetimes shows that static quenching occurs in both of these dual-labeled probes and that it is the dominant quenching mechanism in the Cy3.5-BHQ1 probe. Absorption spectra are consistent with the formation of an excitonic dimer, an intramolecular heterodimer between the Cy3.5 fluorophore and the BHQ1 quencher.     

Single-molecule quantum-dot fluorescence resonance energy transfer.

Colloidal semiconductor quantum dots are promising for single-molecule biological imaging due to their outstanding brightness and photostability. As a proof of concept for single-molecule fluorescence resonance energy transfer (FRET) applications, we measured FRET between a single quantum dot and a single organic fluorophore Cy5. DNA Holliday junction dynamics measured with the quantum dot/Cy5 pair are identical to those obtained with the conventional Cy3/Cy5 pair, that is, conformational changes of individual molecules can be observed by using the quantum dot as the donor.     

Quantum dot-conjugated hybridization probes for preliminary screening of siRNA sequences

In the present study, we describe the design and fabrication of quantum dot-conjugated hybridization probes and their application to the development of a comparatively simple and rapid procedure for the selection of highly effective small-interfering RNA (siRNA) sequences for RNA interference (RNAi) in mammalian cells, for example, siRNAs with high accessibility and affinity to the respective mRNA target. A single-stranded siRNA was conjugated with a quantum dot and used as a hybridization probe. The target mRNA was amplified in the presence of Cy5-labeled nucleotides, and Cy5-mRNA served as a hybridization sample. The formation of siRNA/mRNA duplexes during a comparatively short hybridization time (1 h) was used as a criterion for the selection of highly effective, target-specific siRNA sequences. The accessibility and affinity of the siRNA sequence for the target mRNA site were determined by fluorescence resonance energy transfer (FRET) between a quantum dot (donor) and a fluorescent dye molecule (Cy5, acceptor) localized at an appropriate distance from each other when hybridization occurred. The FRET signal was observed only when there was high accessibility between an antisense siRNA and a sense mRNA and did not appear in the case of mismatch siRNAs. Moreover, the amplitude of the FRET signal significantly correlated with the specific effect of siRNA on the expression of the target mRNA and protein, determined in native cells by RT-PCR and immunoblot analysis, respectively.     

Reverse flow-injection manifold for spectrofluorimetric determination of aluminum in drinking water

A simple reverse flow-injection (rFIA) manifold for the direct determination of aluminum in drinking water is proposed. This rapid and sensitive method is based on the formation of an Al(3+) complex with salicylaldehyde picolinoylhydrazone (SAPH), which shows a maximum blue-green fluorescence (lambda(ex)=384 nm, lambda(em)= 468 nm) at pH 5.4. Operative conditions both for batch and rFIA procedures were investigated including reagent concentration, buffer solutions, injection loop, reacting coil and wavelengths used for the fluorimetric detection. The tolerance limits of foreign ions have been also evaluated, before and after the addition of masking agents. The reverse flow-injection procedure allows determination of Al(3+) at ppb level (LOD: 1.9 mug l(-1)) within a working range of 5-30 mug l(-1). The proposed method was successfully employed for the determination of Al(3+) in several commercial drinking, soft drinking (as certified reference material), and tap water samples.     

Global and target analysis of time-resolved fluorescence spectra of Di-9H-fluoren-9-yldimethylsilane: dynamics and energetics for intramolecular excimer formation

Dynamics and energetics for intramolecular excimer formation of a diarylsilane, di-9H-fluoren-9-yldimethylsilane (DFYDMS) have been investigated by means of ps time-resolved fluorescence spectroscopy and ab initio calculation. Multiple fluorescence decay curves were globally deconvolved to generate time-resolved fluorescence spectra and decay-associated spectra (DAS), from which species-associated spectra (SAS) were obtained. It is shown in the global analysis that there are at least three excited states: Two states are the locally excited (LE) states (lambda(max) approximately 320 nm) having lifetimes of 0.70 +/- 0.04 and 1.75 +/- 0.02 ns, and another is the excimer state (lambda(max) approximately 400 nm) having a lifetime of 7.34 +/- 0.02 ns. The species which decays with 0.70 ns evolves into a species with a red-shifted spectrum, which in turn decays in 7.34 ns. The experimental and ab initio results indicate that the rise time of 0.70 ns corresponds to the conversion of the initial S(1) LE state having a near sandwich geometry to the S(1) excimer state adopting a true sandwich geometry.     

Highly sensitive FRET-based fluorescence immunoassay for aflatoxin B1 using cadmium telluride quantum dots

We report on a competitive immunoassay for the determination of aflatoxin B1 using fluorescence resonance energy transfer (FRET) from anti-aflatoxin B1 antibody (immobilized on the shell of CdTe quantum dots) to Rhodamine 123 (Rho 123-labeled aflatoxin B1 bound to albumin). The highly specific immunoreaction between the antibody against aflatoxin B1 on the QDs and the labeled-aflatoxin B1 brings the Rho 123 fluorophore (acting as the acceptor) and the QDs (acting as the donor) in close spatial proximity and causes FRET to occur upon photoexcitation of the QDs. In the absence of unlabeled aflatoxin B1, the antigen-antibody complex is stable, and strong emission resulting from the FRET from QDs to labeled aflatoxin B1 is observed. In the presence of aflatoxin B1, it will compete with the labeled aflatoxin B1-albumin complex for binding to the antibody-QDs conjugate so that FRET will be increasingly suppressed. The reduction in the fluorescence intensity of the acceptor correlates well with the concentration of aflatoxin B1. The feasibility of the method was established by the detection of aflatoxin B1 in spiked human serum. There is a linear relationship between the increased fluorescence intensity of Rho 123 with increasing concentration of aflatoxin B1 in spike human serum, over the range of 0.1–0.6 μmol·mL^?1. The limit of detection is 2?×?10^?11 M. This homogeneous competitive detection scheme is simple, rapid and efficient, and does not require excessive washing and separation steps.

Porphyrin, phthalocyanine and porphyrazine derivatives with multifluorenyl substituents as efficient deep-red emitters

The synthesis and photophysical properties are described for a series of porphyrin, phthalocyanine and pyrazinoporphyrazine derivatives which bear four or eight peripheral fluorenyl substituents as antennae. Representative examples are 5,10,15,20-tetra(9,9-dihexyl-9H-fluoren-2-yl)porphyrin (2), 5,10,15,20-tetrakis[4-(9,9-dihexyl-9H-fluoren-2-yl)phenyl]porphyrin (3), 2,3,9,10,16,17,23,24-octakis(9,9-dihexyl-9H-fluoren-2-yl)-29H,31H-phthalocyanine (8) and 2,3,9,10,16,17,23,24-octakis[4-(9,9-dihexyl-9H-fluoren-2-yl)phenyl]-29H,31H-tetrapyrazinoporphyrazine (9). Palladium-mediated Suzuki-Miyaura cross-coupling reactions have been key steps for attaching the substituents. The compounds are deep-red emitters: lambda(max)(em)=659 (3), 737 (8) and 684 nm (9). Their absorption and emission spectra, their fluorescence lifetimes and quantum yields are correlated with the structures of the macrocycles and the substituents. The solution fluorescence quantum yields of porphyrin derivatives substituted with fluorene (2-4) and terphenyl substituents (7) (Phi(f)=0.21-0.23) are approximately twice that of tetraphenylporphyrin. For phthalocyanine derivative 8, Phi(f) was very high (0.88). Specific excitation of the fluorene units of 8 produced emission from both of them (lambda(max)=480 nm) and also from the phthalocyanine core (lambda(max)=750 nm), indicating a competitive rate of energy transfer and radiative decay of the fluorenes. Organic light-emitting devices (OLEDs) were made by spin-coating techniques by using a polyspirobifluorene (PSBF) copolymer as the host blended with 3 (5 wt. %) in the configuration ITO/PEDOT:PSS/PSBF copolymer:3/Ca/Al. Deep-red emission (lambda(max)=663 nm; CIE coordinates x=0.70, y=0.27) was observed with an external quantum efficiency of 2.5 % (photons/electron) (at 7.5 mA cm(-2)), a low turn-on voltage and high emission intensity (luminance) of 5500 cd m(-2) (at 250 mA/ m(2)).     

Intermolecular energy transfer involving an iridium complex studied by a combinatorial method

A recently developed combinatorial method utilizing angular dependence of evaporation rate was used to create compositional spread thin film libraries of Tris(2-pyridin-2-yl-indolizino[3,4,5-ab] isoindole-C(1), N('))iridium(III) [Ir(pin)(3)] and 4,4(')-N,N(')-dicarbazol-biphenyl (CBP) composite, with the molar fraction of Ir(pin)(3) complex varying in the 0.0003Ir(pin)(3) energy transfer proceeds by the Forster mechanism with the Forster radius of 30 A. The CBPxIr(pin)(3) composite has the highest photoluminescence quantum efficiency approximately 0.95, for chi(Ir(pin)(3) )=0.03 and is characterized by a structured green emission (lambda(max)=538 nm) originating from the ligand-centered (pi-pi(*))(3) state of the Ir(pin)(3) complex. On the contrary, the PL spectra of Ir(pin)(3) bulk are characterized by a weak red emission (lambda(max)=673 nm) attributed to the lowest metal-to-ligand charge transfer state. A statistical analysis based on a binomial distribution indicates that the emission from the (pi-pi(*))(3) state is quenched in Ir(pin)(3) molecules that are in a direct contact with each other.