Chaoqing Dong

Single-molecule technology for rapid detection of DNA hybridization based on resonance light scattering of gold nanoparticles.

Detection of specific nucleic-acid sequences has important ACHTUNGTRENNUNGapplication in clinical diagnosis, food and drug safety, environmental monitoring, and antibioterrorism. Recently, specific DNA sequence detection techniques based on nanomaterials have offered significant advantages over conventional assay systems because they are label-free (fluorescent or radioactive), PCR-free, and low cost. Gold nanoparticles (GNPs) have been regarded as ideal nanoprobes with potential applications in diagnosis and bioassay development due to their unique optical, physical, and chemical properties. 4] Based on the distancedependent optical properties of cross-linked GNPs during DNA hybridization, Mirkin and co-workers reported a novel colormetric method for the detection DNA-sequence changes in vitro and in vivo. More recently, some assembling technologies, such as colorimetric scatter, hyper Rayleigh scattering light,and linear light scattering have been used to further improve the detection limit. Recently, confocal correlation spectroscopy (CCS) was developed for measuring the particle size of fluorescent quantum dots and nonfluorescent nanoparticles. The setup was similar to fluorescence correlation spectroscopy (FCS), and the detection of nonfluorescent nanoparticles was based on their light scattering. However, it can be difficult for this technology to detect smaller-sized particles since the light-scattering signal is dependent on the size of nanoparticles. Resonance light scattering (RLS) is a very sensitive and selective technology widely used to investigate aggregation and interaction of biomolecules and nanoparticles. GNPs show extremely strong light scattering at the plasmon-resonance wavelength due to the collective oscillation of their conduction electrons. Their RLS signal is orders of magnitude greater than that of nonmetallic objects of the same size, and more than a million-fold that of fluorescent molecules. This strong RLS of GNPs could become fundamental for developing single-molecule technologies. To the best of our knowledge, single-molecule technologies based on the principle of RLS by using GNPs have not been developed to date. Here we describe for the first time a single-molecule detection method, named resonance light scattering correlation spectroscopy (RLSCS), based on the strong RLS of GNPs. This method was successfully used to characterize the size of GNPs and to rapidly analyze solution-phase DNA hybridization. The principle and setup of RLSCS (see Figure S1 in the Supporting Information) is analogous to that of FCS. In RLSCS the RLS intensity of GNPs, which arises from a very small illuminated volume (<1 fL), is correlated to obtain information about the processes that generate fluctuations in RLS intensity of nanoparticles. The sample volume (0.9 fL in this study) is defined by a highly focused laser beam and a pinhole. The scattering light is collected with a high numerical aperture microscope objective and monitored by a sensitive single-photon counting device. The measured scattering light fluctuates with time and the temporal fluctuations are autocorrelated. Like FCS, RLSCS can be used to obtain scattering-light intensities and diffusion time of GNPs, as well as other information calculated by using an autocorrelation function. First, we used the home-built RLSCS system to characterize the radii of colloidal GNPs based on their characteristic diffusion time and Stokes–Einstein equation. Excellent autocorrelation curves were obtained for four GNP samples (Figure 1A); these were similar to the FCS curves. The experimental data were well fitted with the theoretical autocorrelation function, G(t). In addition, the hydrodynamic diameters of the four samples were found to be 19, 24, 58, and 91 nm; these data were in good agreement with the results (21, 25, and 53 nm) obtained from transmission electron microscopy (TEM) for the small diameter ranges of GNPs (Figure S3). However, bigger GNPs (91 nm hydrodynamic diameter) considerably deviated from the results of the TEM experiments (75 nm; Figure 1A inset) ; this was mainly due to optical trapping of large GNPs in the highly ACHTUNGTRENNUNGfocused laser beam, and the wider size distribution of the GNPs. The results suggest that low power of light should be used in characterization of bigger GNPs so as to reduce the ACHTUNGTRENNUNGeffects of optical trapping. The count-rate traces showed that there was an approximately 60-fold increase in scattering-light intensity from 21 to 75 nm GNPs (Figure 1B); this illustrates that scattering-light ACHTUNGTRENNUNGintensity was strongly dependent on the size of nanoparticle. Moreover, the surface-plasmon band of GNPs (Figure S2B) slightly red-shifted with an increase in particle size. The resonance-scattering peak of GNPs (Figure S2C) was located at about 620 nm, which was close to the wavelength of the He– Ne laser (632.8 nm) used in our experiments; this might be the major reason for the very strong RLS signal. These data also demonstrated that light scattering of GNPs in solution was associated with the wavelength of the incident light. Based on our experience with FCS, the RLS signal from a single GNP (21 nm) was ten-times higher than that from a single fluorescent molecule (Alexa Fluor 647). Interestingly, the autocorrelation curves of unmodified GNPs displayed two stages. The first stage (timescale from 1 to 200 ms) was attributed to the rotational diffusion of GNPs, [a] K. Wang, X. Qiu, C. Dong, Prof. Dr. J. Ren College of Chemistry and Chemical Engineering Shanghai Jiaotong University 800 Dongchuan Road, Shanghai, 200240 (China) Fax: (+86)21-54741297 E-mail : jicunren@sjtu.edu.cn Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.

Single gold nanoparticles counter: an ultrasensitive detection platform for one-step homogeneous immunoassays and DNA hybridization assays.

In this paper, we present for the first time a single gold nanoparticle counter (SGNPC) in solution based on the photon bursting in a highly focused laser beam (less than 1 fL) due to the plasmon resonance scattering and Brownian motion of gold nanoparticles (GNPs). The photon burst intensity of single 36 nm GNPs is several tens to hundreds times stronger than that of quantum dots (QDs) and organic dyes. The relationship between the photon burst counts and GNPs concentration shows an excellent linearity. The linear range is over 4 orders of magnitude, and the detection limit of GNPs (36 nm) is 17 fM. On the basis of this single nanoparticle technique, we developed an ultrasensitive and highly selective detection platform for homogeneous immunoassay and DNA hybridization assays using GNPs as probes, which were 2-5 orders of magnitude more sensitive than current homogeneous methods. We used this technology to construct homogeneous sandwich immunoassays for cancer biomarkers, such as carcinoembryonic antigen (CEA) and alpha fetal protein (AFP), and aptamer recognition for thrombin. The detection limits are 130 fM for CEA, 714 fM for AFP and 2.72 pM for thrombin. Our method was successfully applied for direct determination of CEA, AFP and thrombin levels in sera from healthy subjects and cancer patients. In homogeneous DNA hybridization detection, we chose methylenetetrahydrofolate reductase (MTHFR) gene as a target. This assay successfully distinguished DNA sequences with single base mismatches, and the detection limits for the target were at 1 fM level.

Sizes of water-soluble luminescent quantum dots measured by fluorescence correlation spectroscopy

In this paper, fluorescence correlation spectroscopy (FCS) was applied to measure the size of water-soluble quantum dots (QDs). The measurements were performed on a home-built FCS system based on the Stokes-Einstein equation. The obtained results showed that for bare CdTe QDs the sizes from FCS were larger than the ones from transmission electron microscopy (TEM). The brightness of QDs was also evaluated using FCS technique. It was found that the stability of the surface chemistry of QDs would be significantly improved by capping it with hard-core shell. Our data demonstrated that FCS is a simple, fast, and effective method for characterizing the fluorescent quantum dots, and is especially suitable for determining the fluorescent nanoparticles less than 10nm in water solution.

Highly sensitive homogenous immunoassay of cancer biomarker using silver nanoparticles enhanced fluorescence correlation spectroscopy.

In this paper, we developed a highly sensitive homogeneous immunoassay by combining fluorescence correlation spectroscopy (FCS) with silver nanoparticles (SNPs)-antibody conjugates as probes. We first synthesized 14nm SNPs in aqueous solution and then modified SNPs with 11-mercaptoundecanoicacid (MUA) via SNP-S bond. Resonance light scattering correlation spectroscopy (RLSCS) was utilized to characterize SNPs and MUA-functionalized SNPs (MUA-SNPs). The immune reaction of alpha fetal protein (AFP) antigen and its antibody was used as a reaction model and AFP labeled with Alexa Fluor 647 was used as the tracer antigen in homogeneous competitive immunoassay. We observed that the antigen-antibody complexes showed the significant increase in the diffusion times and fluorescence intensity compared to free dye-labeled antigen. On the advantages of the effects of SNPs on fluorescence enhancement and diffusion time, the homogeneous competitive immunoassay was performed by the two-component model analysis of FCS. Under the optimal condition, the detection limit was 1.5pM and the linear range was from 6pM to 60pM (R>0.99). This assay was successfully applied for the determination of the AFP level in human serum samples, the relative standard deviation was about 5%, and the recoveries were over 90%.

Aqueous synthesis of CdTe/CdS/ZnS quantum dots and their optical and chemical properties

In this paper, we described a strategy for synthesis of thiol-coated CdTe/CdS/ZnS (core-shell-shell) quantum dots (QDs) via aqueous synthesis approach. The synthesis conditions were systematically optimized, which included the size of CdTe core, the refluxing time and the number of monolayers and the ligands, and then the chemical and optical properties of the as-prepared products were investigated. We found that the mercaptopropionic acid (MPA)-coated CdTe/CdS/ZnS QDs presented highly photoluminescent quantum yields (PL QYs), good photostability and chemical stability, good salt tolerance and pH tolerance and favorable biocompatibility. The characterization of high-resolution transmission electron microscopy (HRTEM), X-ray powder diffraction (XRD) and fluorescence correlation spectroscopy (FCS) showed that the CdTe/CdS/ZnS QDs had good monodispersity and crystal structure. The fluorescence life time spectra demonstrated that CdTe/CdS/ZnS QDs had a longer lifetime in contrast to fluorescent dyes and CdTe QDs. Furthermore, the MPA-stabilized CdTe/CdS/ZnS QDs were applied for the imaging of cells. Compared with current synthesis methods, our synthesis approach was reproducible and simple, and the reaction conditions were mild. More importantly, our method was cost-effective, and was very suitable for large-scale synthesis of CdTe/CdS/ZnS QDs for future applications.

Single particle technique for one-step homogeneous detection of cancer marker using gold nanoparticle probes

In this paper, we reported a single particle technique for the one-step homogeneous immunoassay of a cancer marker by resonance light scattering correlation spectroscopy (RLSCS). The setup of RLSCS was similar to fluorescence correlation spectroscopy (FCS), and its principle was based on measuring the resonance light scattering fluctuations in a small volumes (less than 1 fL) due to Brownian motion of single particles. In homogeneous immunoassay, we used a sandwich strategy and conjugated two different antibodies (Ab) with gold nanoparticles (GNPs) respectively. When two different GNPs labeled with antibodies are mixed in a sample containing antigen (Ag) targets, the binding of targets will cause GNPs to form dimers (or oligomers), which leads to the significant increase in the characteristic diffusion time of GNPs in the detection volume. The RLSCS method can sensitively detect the change in the characteristic diffusion time of GNPs before and after immune reactions. We used this technology in homogeneous immunoassays for the liver cancer biomarker alpha-fetoprotein (AFP). The conditions of the immune reaction were investigated systematically. In the optimal conditions, the linear range of this assay is from 1 pM to 1 nM and the detection limit is 1 pM for AFP. This new method was successfully applied for the direct determination of AFP levels in sera from healthy subjects and cancer patients. Our results were in good agreement with ELISA assays.

Tempo-spatially resolved scattering correlation spectroscopy under dark-field illumination and its application to investigate dynamic behaviors of gold nanoparticles in live cells.

In this study, a new tempo-spatially resolved fluctuation spectroscopy under dark-field illumination is described, named dark-field illumination-based scattering correlation spectroscopy (DFSCS). DFSCS is a single-particle method, whose principle is similar to that of fluorescence correlation spectroscopy (FCS). DFSCS correlates the fluctuations of the scattered light from single nanoparticle under dark-field illumination. We developed a theoretical model for translational diffusion of nanoparticles in DFSCS system. The results of computer simulations documented that this model was able to well describe the diffusion behaviors of nanoparticles in uniformly illuminated field. The experimental setup of DFSCS was achieved by introducing a dark-field condenser to the frequently used bright-field microscope and an electron multiplying charge-coupled device (EMCCD) as the array detector. In the optimal condition, a stack of 500 000 frames were collected simultaneously on 64 detection channels for a single measurement with acquisition rate of 0.5 ms per frame. We systematically investigated the effect of certain factors such as particle concentration, viscosity of the solution, and heterogeneity of gold nanoparticles (GNPs) samples on DFSCS measurements. The experiment data confirmed theoretical model proposed. Furthermore, this new method was successfully used for investigating dynamic behaviors of GNPs in live cells. Our preliminary results demonstrate that DFSCS is a practical and affordable tool for ordinary laboratories to investigate the dynamic information of nanoparticles in vitro as well as in vivo.

Non-blinking (Zn)CuInS/ZnS Quantum Dots Prepared by In Situ Interfacial Alloying Approach

Semiconductor quantum dots (QDs) are very important optical nanomaterials with a wide range of potential applications. However, blinking behavior of single QD is an intrinsic drawback for some biological and photoelectric applications based on single-particle emission. Herein we present a rational strategy for fabrication of non-blinking (Zn)CuInS/ZnS QDs in organic phase through in situ interfacial alloying approach. This new strategy includes three steps: synthesis of CuInS QDs, eliminating the interior traps of QDs by forming graded (Zn)CuInS alloyed QDs, modifying the surface traps of QDs by introducing ZnS shells onto (Zn)CuInS QDs using alkylthiols as sulfur source and surface ligands. The suppressed blinking mechanism was mainly attributed to modifying QDs traps from interior to exterior via a step-by-step modification. Non-blinking QDs show high quantum yield, symmetric emission spectra and excellent crystallinity, and will enable applications from biology to optoelectronics that were previously hindered by blinking behavior of traditional QDs.

Sensitive single particle method for characterizing rapid rotational and translational diffusion and aspect ratio of anisotropic nanoparticles and its application in immunoassays.

In this article, we reported a new and sensitive method for characterizing rapid rotational and translational diffusion of gold nanoparticles (GNPs) and gold nanorods (GNRs) by resonance light scattering correlation spectroscopy (RLSCS). The RLSCS is a new single nanoparticle method, and its principle is based on measuring the resonance light scattering fluctuations in a highly focused volume due to Brownian motion of single particles, which resembles fluorescence correlation spectroscopy (FCS). On the basis of the theory of FCS, we first developed a model for rotational and translational diffusion and aspect ratio of nanoparticles in the RLSCS system. Then, we investigated the effects of certain factors such as the wavelength of illumination light and viscosity of solution using GNPs and GNRs as model samples and discovered that the polarization anisotropy and the scattering light intensity of GNPs and GNRs were significantly dependent on the wavelengths of illumination light. Using the 632.8 nm He-Ne laser as a light source, which was close to the resonance scattering band, we successfully obtained the translational and rotational diffusion coefficients and aspect ratios of anisotropic nanoparticles by the RLSCS method. The results obtained by this new method were in good agreement with transmission electron microscopy and theoretical calculation. Furthermore, the homogeneous sandwich immunoreaction was investigated using the antibody-modified GNPs as the probes. The changes in translational diffusion behaviors and aspect ratios of GNPs in immunoreaction were observed by the RLSCS method. By these changes, we can develop a new homogeneous immunoassay. Our preliminary results illustrated that the RLSCS method was a powerful tool for characterizing rapid rotational and translational diffusion behaviors of anisotropic nanoparticles in solution. We believe that the RLSCS method exhibits the wide applications in biological science especially in vivo study on the interaction of nanoparticles and biomolecules.

Ultrahighly sensitive homogeneous detection of DNA and microRNA by using single-silver-nanoparticle counting.

DNA and RNA analysis is of high importance for clinical diagnoses, forensic analysis, and basic studies in the biological and biomedical fields. In this paper, we report the ultrahighly sensitive homogeneous detection of DNA and microRNA by using a novel single-silver-nanoparticle counting (SSNPC) technique. The principle of SSNPC is based on the photon-burst counting of single silver nanoparticles (Ag NPs) in a highly focused laser beam (about 0.5 fL detection volume) due to Brownian motion and the strong resonance Rayleigh scattering of single Ag NPs. We first investigated the performance of the SSNPC system and then developed an ultrasensitive homogeneous detection method for DNA and microRNA based on this single-nanoparticle technique. Sandwich nucleic acid hybridization models were utilized in the assays. In the hybridization process, when two Ag-NP-oligonucleotide conjugates were mixed in a sample containing DNA (or microRNA) targets, the binding of the targets caused the Ag NPs to form dimers (or oligomers), which led to a reduction in the photon-burst counts. The SSNPC method was used to measure the change in the photon-burst counts. The relationship between the change of the photon-burst counts and the target concentration showed a good linearity. This method was used for the assay of sequence-specific DNA fragments and microRNAs. The detection limits were at about the 1 fM level, which is 2-5 orders of magnitude more sensitive than current homogeneous methods.