Co-reporter:Di Su, Xiaocai Hu, Chaoqing Dong, and Jicun Ren
Analytical Chemistry September 19, 2017 Volume 89(Issue 18) pp:9788-9788
Publication Date(Web):August 14, 2017
DOI:10.1021/acs.analchem.7b01735
Caspase-3 is a key enzyme executing apoptosis during ontogenesis and homeostasis of multicellular organisms, and is a very important and potential drug target in treatment of apoptosis disturbance. So far, no commercial drugs for caspase-3 are available, and it is urgently necessitated to develop an effective method for caspase-3 activity assay and its inhibitor screening. In this paper, we propose a new method for determination of caspase-3 activity and its inhibition constant by combining single molecule fluorescence correlation spectroscopy (FCS) with a microwell chip. Its principle is based on measurement of the enzyme reaction kinetics and homogeneous detection of the reaction product by FCS. This system can reduce the requirement sample volume to 1 μL level. The caspase-3 substrates are doubly labeled with fluorophore and biotin, the enzyme reaction can be quickly terminated in the presence of streptavidin, and the reaction products can be selectively detected by FCS. We established the model of caspase-3 inhibitor screening by combining the dynamics of enzyme reaction with FCS theory. This new method was successfully used for determination of inhibition constants of certain inhibitors and assay of drug-induced apoptosis. Compared to current methods, this method shows high sensitivity, small reagent dosage and short analysis time. We believe that this method will become an efficient platform for screening of caspase-3 inhibitors and detection of apoptosis.
Co-reporter:Bocheng Zhang, Heng Liu, Xiangyi Huang, Chaoqing Dong, and Jicun Ren
Analytical Chemistry November 21, 2017 Volume 89(Issue 22) pp:12609-12609
Publication Date(Web):October 27, 2017
DOI:10.1021/acs.analchem.7b04166
A single-nanoparticle detection method is reported for characterizing the size distribution of noble metal nanoparticles in solution by combining resonance light scattering correlation spectroscopy (RLSCS) with the maximum entropy method (MEM). The principle of RLSCS is based on the autocorrelation analysis of the resonance light scattering (RLS) fluctuations due to Brownian motion of a single nanoparticle in a highly focused detection volume (less than 1.0 fL), which resembles fluorescence correlation spectroscopy (FCS). However, RLS intensity of nanoparticles such as gold nanoparticles (GNPs) is proportional to the sixth power of sizes according to the Mie theory, which is different from the optical properties of fluorescent molecules. Herein the present FCS theoretical model cannot be directly applied in RLSCS to characterize GNPs. In this study, we used GNPs as model samples and first established an RLSCS theoretical model for the size distribution of GNPs by using the maximum entropy method (MEM), which is called MEM-RLSCS. This model covers the contribution of single-particle brightness of GNPs to the MEM fitting process based on the Mie theory. Then we preformed computer simulations of this model. The simulated results documented that the model proposed was able to well describe the diffusion behaviors and size distribution of nanoparticles. We investigated the effects of certain factors such as size difference, the relative concentration, and single-particle brightness on the size distribution. Finally, we used the MEM-RLSCS for characterization of GNPs in solution, and the results obtained were in agreement with the size distribution of GNPs from transmission electron microscopy (TEM). This method is also suitable for characterization of other metal nanoparticles (such as silver nanoparticles) in solution and in situ study diffusion dynamics of nanoparticles in living cells.
Co-reporter:Jinjie Wang, Xiangyi Huang, Heng Liu, Chaoqing Dong, and Jicun Ren
Analytical Chemistry May 16, 2017 Volume 89(Issue 10) pp:5230-5230
Publication Date(Web):April 24, 2017
DOI:10.1021/acs.analchem.6b04547
In this work, we propose fluorescence and scattering light cross-correlation spectroscopy (FSCCS) based on laser confocal configuration using silver nanoparticle (SNPs) and Alexa Fluor 488 (Alexa) as probe pairs. FSCCS is a single molecule (particle) method, and its principle is similar to that of fluorescence cross-correlation spectroscopy (FCCS). We established the setup of FSCCS using single wavelength laser and developed an immunoassay model of FSCCS. The reliability and adaptability of FSCCS method were evaluated by homogeneous sandwich immunoassay mode. In the study, liver cancer biomarker alpha-fetoprotein (AFP) was used as an assay model, two different antibodies were labeled with SNPs and fluorophore Alexa Fluor 488, respectively. In the optimal conditions, the linear range of AFP covers 5 pM to 580 pM and the detection limit is 3.1 pM. This method was successfully applied for direct determination of AFP levels in human serum samples, and the obtained results were in good agreement with data obtained via ELISAs. The advantage of this method lies in its simplicity, attractive SNPs probes, high sensitivity and selectivity and high efficiency. We believe that FSCCS method exhibits promising potential applications in homogeneous bioassays and study on the molecular interaction and nanoparticle-molecule interaction.
Co-reporter:Aidi Zhang, Yannan Bian, Jinjie Wang, Kuiyong Chen, Chaoqing Dong and Jicun Ren
Nanoscale 2016 vol. 8(Issue 9) pp:5006-5014
Publication Date(Web):28 Jan 2016
DOI:10.1039/C5NR08504G
Semiconductor quantum dots (QDs) are very important fluorescent nanocrystals with excellent optical properties. However, QDs, at the single-particle level, show severe fluorescence intermittency (or blinking) on a wide time scale from milliseconds to minutes, which limits certain optical and biological applications. Generally, blinking behavior of QDs strongly depends on their surface state and surrounding environment. Therefore, current blinking suppression approaches are mostly focused on the introduction of an inorganic shell and organic small molecule compounds. In this study, we described a “bottom up” approach for the synthesis of CdSe/CdS/polymer core/shell/shell QDs via the in situ one-pot polymerization approach in order to control the blinking behavior of QDs. Three monomers (dithiothreitol (DTT), phenylenediamine (PDA), and hexamethylenediamine (HDA)) were respectively used to polymerize with hexachlorocyclotriphosphazene (HCCP), and then the polyphosphazene polymers were obtained with cyclotriphosphazene as the basic macromolecular backbone. By regulating the molar ratios of the activated comonomers, we can control the blinking behavior of CdSe/CdS/polymer QDs. Under the optimal conditions, the percentage of “non-blinking” CdSe/CdS/polymer QDs (the “on time” fraction > 99% of the overall observation time) was up to 78%. The suppression mechanism was attributed to the efficient passivation of QD surface traps by the sulfhydryl or phenyl groups in the polyphosphazene polymers.
Co-reporter:Xiaocai Hu;Di Su;Zhixue Du;Xiangyi Huang;Jicun Ren
Microchimica Acta 2016 Volume 183( Issue 8) pp:2457-2465
Publication Date(Web):2016 August
DOI:10.1007/s00604-016-1891-7
We report on a method for direct determination of the molar concentration of gold nanoparticles (GNP) in solution by using resonance light scattering correlation spectroscopy (RLSCS). RLSCS is based on the correlation analysis of the fluctuations of resonance light scattering due to Brownian motion of single nanoparticles in a highly-focused laser volume. Similar to single molecule fluorescence correlation spectroscopy, the number of particles in the detection volume is reciprocally related to the G(0) value in the RLSCS plot. A model is established for quantification of GNP concentration, and the effect of laser illumination intensity was studied. An excellent linear relationship exists between the concentration of GNP and the reciprocal G(0) value of the correlation plots at low laser illumination intensity. The method was applied to the determination of the molar concentrations of GNP in sizes of 15, 20, 30, and 40 nm to give detection limits of 300, 80, 10, and 40 pM, respectively. The results obtained by RLSCS were in good agreement with that of combined atomic emission spectroscopy and transmission electron microscopy (AES-TEM). A sensitive microscale method was reported for the determination of the activity of the enzyme caspase 3 by RLSCS by using GNP as labeling probes. The assay is based on the measurement of the change of the molar concentration of GNP when peptide-labeled GNP substrates were cleaved by caspase 3. The method is shown to enable the determination of caspase 3 (with a 0.1 nM detection limit) and to monitoring drug-induced cell apoptosis.
Co-reporter:Zhancheng Xu;Tao Lan;Xiangyi Huang;Jicun Ren
Luminescence 2015 Volume 30( Issue 5) pp:605-610
Publication Date(Web):
DOI:10.1002/bio.2793
Abstract
We described a new and sensitive method for the determination of mercury ions (Hg2+) on the basis of fluorescence correlation spectroscopy (FCS) and recognition of oligonucleotides. In this assay, 30-nm gold nanoparticles (GNPs) were modified with oligonucleotides containing thymine bases (T) as fluorescent probes, and the principle of this assay was based on the specific binding of Hg2+ by two DNA thymine bases. When two GNPs labelled with different oligonucleotides were mixed with a sample containing Hg2+, the T-Hg2+-T binding reaction should cause GNPs to form dimers (or oligomers), which would lead to a significant increase in the characteristic diffusion time of GNPs in the detection volume. The FCS method is a single molecule detection method and can sensitively detect the change in the characteristic diffusion time of GNPs before and after binding reactions. The quantitative analysis was performed according to the relation between the change in the characteristic diffusion time of GNPs and the concentration of Hg2+. Under optimal conditions, the linear range of this method was from 0.3 nM to 100 nM, and the detection limit was 0.14 nM for Hg2+. This new method was successfully applied for direct determination of Hg2+ levels in water and cosmetics samples. Copyright © 2014 John Wiley & Sons, Ltd.
Co-reporter:Bocheng Zhang ; Tao Lan ; Xiangyi Huang ; Chaoqing Dong ;Jicun Ren
The Journal of Physical Chemistry C 2014 Volume 118(Issue 26) pp:14495-14501
Publication Date(Web):June 11, 2014
DOI:10.1021/jp500843k
In this work, we reported an efficient method for eliminating the optical trapping effect on characterization of nanoparticle diffusion parameters by resonance light scattering correlation spectroscopy (RLSCS). The RLSCS represents a new single nanoparticle method and its principle was based on measuring the resonance light scattering fluctuations in a highly focused laser beam due to the Brownian motion of single nanoparticles such as gold nanoparticles (GNPs), which resembled fluorescence correlation spectroscopy (FCS). In RLSCS analysis, the polarizability of nanoparticles are much higher than fluorescent molecules in FCS, and the sizes of them are larger, therefore, the optical trapping force significantly affects the diffusion behaviors of nanoparticles under a highly focused laser beam. In this study, we used the 632.8 nm He—Ne laser as the light source, which was close to the resonance scattering band of GNPs, and chose GNPs (from 20 to 100 nm) as model samples. We theoretically and experimentally investigated the optical trapping effect of GNPs in RLSCS, and observed a good linear relation between the characteristic diffusion times of GNPs and laser intensity in the certain condition (below 100 μW). This result was in line with the theoretical deduction. By the extrapolation strategy, we effectively eliminated the optical trapping effect and accurately obtained the diameter of GNPs, which was in good agreement with that obtained by transmission electron microscopy. The method described here can extend to FCS analysis of fluorescent nanoparticles as well.
Co-reporter:Chaoqing Dong, Heng Liu, and Jicun Ren
Langmuir 2014 Volume 30(Issue 43) pp:12969-12976
Publication Date(Web):2017-2-22
DOI:10.1021/la503055v
The current method for investigating the blinking behavior is to immobilize quantum dots (QDs) in the matrix and then apply a fluorescent technique to monitor the fluorescent trajectories of individual QDs. So far, no method can be used to directly assess the blinking state of ensemble QDs in free solution. In this study, a new method was described to characterize the blinking state of the QDs in free solution by combining single molecule fluorescence correlation spectroscopy (FCS) with ensemble spectroscopic methods. Its principle is based on the observation that the apparent concentration of bright QDs obtained by FCS is less than its actual concentration measured by ensemble spectroscopic method due to the QDs blinking. We proposed a blinking index (Kblink) for characterizing the blinking state of QDs, and Kblink is defined as the ratio of the actual concentration (Cb,actual) measured by the ensemble spectroscopic method to the apparent concentration (Cb,app) of QDs obtained by FCS. The effects of certain factors such as laser intensity, growth process, and ligands on blinking of QDs were investigated. The Kblink data of QDs obtained were successfully used to characterize the blinking state of QDs and explain certain experimental results.
Co-reporter:Dr. Chaoqing Dong;Heng Liu;Aidi Zhang ;Dr. Jicun Ren
Chemistry - A European Journal 2014 Volume 20( Issue 7) pp:1940-1946
Publication Date(Web):
DOI:10.1002/chem.201303605
Abstract
Semiconductor quantum dots (QDs) are very important optical nanomaterials with a wide range of potential applications. However, the blinking of single QDs is an intrinsic drawback for some biological and photoelectric applications based on single-dot emission. In this work, we systematically investigated the effects of certain synthetic conditions on the blinking behavior of aqueous CdTeS alloyed QDs, and observed that blinking behaviors of QDs were able to be controlled by the structure and concentration of the thiol compounds that were used as surface ligands. In optimal conditions, completely nonblinking QDs were prepared using certain thiol ligands as stabilizers in aqueous phase. The suppressed blinking mechanism was mainly attributed to elimination of QDs surface traps by coordination of thiol ligands with vacant Cd atoms, formation of appropriate CdS coating on QDs, and controlling the growth dynamics of QDs. Nonblinking QDs show high quantum yield, small size, and good solubility, and will be applied to some fields that were previously limited by blinking of traditional QDs.
Co-reporter:Jialing Li, Chaoqing Dong, Jicun Ren
TrAC Trends in Analytical Chemistry (April 2017) Volume 89() pp:181-189
Publication Date(Web):April 2017
DOI:10.1016/j.trac.2017.02.004
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