Letter pubs.acs.org/ac
Simple and Accurate Quantification of Quantum Yield at the SingleMolecule/Particle Level Juan Hu and Chun-yang Zhang* Single-Molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Guangdong 518055, China S Supporting Information *
ABSTRACT: Quantum yield represents one of most fundamental and important properties of photoluminescent materials and eventually determines the suitability of materials for the applications in optical devices, analysis, biosensing, and fluorescence imaging. Despite that a variety of methods have been developed to measure the quantum yield, its accurate quantification still remains a great challenge. Here, we develop a new approach with the capability to quantify the quantum yield at the single-molecule/particle level. The quantum yield obtained by the single-molecule/ particle detection is in agreement with that obtained by the conventional optical method. Importantly, this method can accurately measure not only the quantum yield of organic dyes but also that of quantum dots and can be further extended to measure the quantum yield of various fluorescent materials, including the nontransparent sample whose quantum yield is impossible to obtain using the conventional optical method.
F
low cost.2,6,11−13 However, the conventional optical method is established for the simplest case of transparent solution with the involvement of high-concentration bulk solution for both absorption and emission spectral measurement.1,2,6,13 In the conventional optical method, the quantum yield (Q) of a fluorophore is calculated on the basis of eq 2:2,11−13
or any photoluminescent species, quantum yield represents one of most fundamental and important properties.1 Quantum yield is usually used to characterize the photoluminescent species, evaluate the extent of internal conversion and intersystem crossing, and determine the purity of materials.2,3 In theory, the quantum yield (Q) of a photoluminescent molecule is defined as the efficiency of the conversion of absorbed photons (Nabs) to emitted photons (Nem):2−4
Q=
Nem Nabs
Q = Qr ×
(2)
where Q is the quantum yield, I is the integrated intensity of the emission spectra, A is the absorbance at the excitation wavelength, n is the refractive index, and the subscript r refers to the reference fluorophore with a known quantum yield. It should be noted that in the conventional optical method the quantification of quantum yield is usually performed in the bulk solution where the measured total number of emitted photons which escape the bulk sample is not the same as emitted photons in eq 1.2 Moreover, the existence of collisional quenching,2,3 reabsorption,2,3,6−8,13 formation of nonemissive aggregates,8,9 and the inner filter effect3,8−13 in the bulk solution may lead to the decrease of actual emitted radiation and the underestimation of quantum yield.2,3,8 Thus, it has been proposed that an efficient way to minimize the errors related to the bulk solution is to work at the lowest concentration possible.2 Single molecule detection techniques have the capability to work at the extremely low concentration, i.e., at the single-molecule level.14 A hallmark of single molecule
(1)
Quantum yield can be obtained by measuring Nem and Nabs absolutely with an integrating sphere setup.5−7 This method can avoid the uncertainties related to the use of inherent fluorescence standards, but it relies on both the expensive instrument and the linearity range of detection system and suffers from reabsorption effect-induced inaccuracy as well.5−7 Alternatively, quantum yield can be obtained indirectly by exploiting the dissipated heat with photoacoustic spectroscopy7,8 and thermal lensing.9,10 These methods require a good and reproducible contact between the sample and acoustic transducer, as well as a close match between the thermal transport coefficiency of sample and that of reference, and suffer from the fluctuations in the incident radiant power and laser excitation-induced photodecomposition as well.7−10 In addition, quantum yield can be determined fluorometrically relative to a fluorescent standard with a known quantum yield,2,6,11−13 which is termed as “the conventional optical method” in this paper. This method is frequently used for the quantification of quantum yield because of its simplicity and © 2013 American Chemical Society
A I n2 × r × 2 Ir A nr
Received: December 16, 2012 Accepted: February 4, 2013 Published: February 4, 2013 2000
dx.doi.org/10.1021/ac3036487 | Anal. Chem. 2013, 85, 2000−2004
Analytical Chemistry
Letter
tracted from each frame before the analysis of single-molecule/ particle fluorescence intensity. The quantum yield at the singlemolecule/particle level was calculated according to eq 3.
detection, in contrast to the ensemble measurement, is the unparalleled capability to obtain the functional form of the distribution of experimental outcomes and not merely their averages.14 The single-molecule detection techniques have provided an alternative way for ultrasensitive detection of various low-abundance biomolecules without the involvement of target application.14 Here, we develop a new approach with the capability to quantify the quantum yield at the singlemolecule/particle level. This method has the significant advantages of high accuracy, simplicity, low sample consumption, and wide applicability as compared with the conventional optical method.
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RESULTS AND DISCUSSION The quantum yield (Q) obtained by the single-molecule/ particle detection can be deduced from eq 2: Q = Qr ×
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= Qr ×
EXPERIMENTAL SECTION Materials. The 605 nm-emission quantum dots (605QDs) and the 655 nm-emission quantum dots (655QDs) were purchased from Invitrogen Co. (Carlsbad, CA, USA). Rhodamine 101 inner salt (R101), Rhodamine 6G (R6G), and polylysine were obtained from Sigma-Aldrich Inc. (Saint Louis, MO, USA). Oxazine 4 perchlorate (LD690) was purchased from J&K Scientific Ltd. (Beijing, China). The glass coverslips were obtained from Thermo Fisher Scientific Inc. (Portsmouth, NH, USA). Quantification of Quantum Yield by the Conventional Optical Method. The absorption spectra were recorded on a PerkinElmer Lambda 25 UV/vis spectrophotometer (PerkinElmer, Waltham, MA, USA) using the sample with a small absorbance of
