Xiangyi Huang

Colloidal Stability of Gold Nanoparticles Modified with Thiol Compounds: Bioconjugation and Application in Cancer Cell Imaging

Gold nanoparticles (GNPs) are attractive alternative optical probes and good biocompatible materials due to their special physical and chemical properties. However, GNPs have a tendency to aggregate particularly in the presence of high salts and certain biological molecules such as nucleic acids and proteins. How to improve the stability of GNPs and their bioconjugates in aqueous solution is a critical issue in bioapplications. In this study, we first synthesized 17 nm GNPs in aqueous solution and then modified them with six thiol compounds, including glutathione, mercaptopropionic acid (MPA), cysteine, cystamine, dihydrolipoic acid, and thiol-ending polyethylene glycol (PEG-SH), via a Au-S bond. We systematically investigated the effects of the thiol ligands, buffer pH, and salt concentrations of the solutions on the colloidal stability of GNPs using UV-vis absorption spectroscopy. We found that GNPs modified with PEG-SH were the most stable in aqueous solution compared to other thiol compounds. On the basis of the above results, we developed a simple and efficient approach for modification of GNPs using a mixture of PEG-SH and MPA as ligands. These biligand-modified GNPs were facilely conjugated to antibody using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and N-hydroxysulfosuccinimide as linkage reagents. We conjugated GNPs to epidermal growth factor receptor antibodies and successfully used the antibody-GNP conjugates as targeting probes for imaging of cancer cells using the illumination of a dark field. Compared to current methods for modification and conjugation of GNPs, our method described here is simple, has a low cost, and has potential applications in bioassays and cancer diagnostics and studies.

Quantum dot-based FRET for sensitive determination of hydrogen peroxide and glucose using tyramide reaction.

In this paper, we report a new strategy for detection of hydrogen peroxide and glucose using quantum dot (QD)-based fluorescence resonance energy transfer (FRET) and tyramide reaction. The principle of FRET is based on highly sensitive reaction of a carbocyanine dye (Cy5) labeled tyramide and hydrogen peroxide catalyzed by horseradish peroxidase (HRP), and the fluorescence spectrum of QDs (EXmax 605 nm) partially overlaps with the absorption bands of Cy5. We firstly conjugated HRP to QDs, and then demonstrated an efficient FRET between HRP conjugated QDs (as energy donors) and tyramide labeled Cy5 (as energy acceptors) due to the formation of Cy5-labeled HRP-QDs assemblies in the presence of H₂O₂. We observed that the fluorescence Cy5 depended linearly on the H₂O₂ concentration within a range of concentration from 10 to 100 nM and the detection limit of this assay was 10 nM. Based on the principle for determination of H₂O₂, we develop a new strategy for assay of glucose by coupling with glucose oxidase-mediated reaction. The established methods were successfully used for determination of glucose levels in human sera, and the results obtained were in good agreement with commercially available method. Our method is at least 1 order of magnitude more sensitive than in the commercially available method. More importantly, our method described here can be extended to other assay designs using different oxidase enzymes, energy donors and energy acceptors, such as near-infrared (NIR)-to-visible upconversion nanoparticles and silicon and carbon QDs.

Studies on bioconjugation of quantum dots using capillary electrophoresis and fluorescence correlation spectroscopy.

In this paper, we systematically investigated the conjugation of quantum dots (QDs) with certain biomolecules using capillary electrophoresis (CE) and fluorescence correlation spectroscopy (FCS) methods. Commercial QDs and aqueous‐synthesized QDs in our lab were used as labeling probes, certain bio‐macromolecules, such as proteins, antibodies, and enzymes, were used as mode samples, and 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N‐hydroxysulfo‐succinimide (Sulfo‐NHS) were used as linking reagents. We studied the effects of certain factors such as the isoelectric points (pIs) of bio‐macromolecules and buffer pH on the bioconjugation of QDs, and found that the pIs of bio‐macromolecules played an important role in the conjugation reaction. By the optimization of the buffer pH some proteins with different pIs were efficiently conjugated with QDs using EDC and Sulfo‐NHS as linking agents. Furthermore, we on‐line investigated the kinetic process of QDs‐bioconjugation by FCS and found that the conjugation reaction of QDs with protein was rapid and the reaction process almost completed within 10 min. We also observed that QDs conjugated with proteins were stable for at least 5 days in phosphate buffer. Our work described here will be very helpful for the improvement of the QDs conjugation efficiency in bioapplications.

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.

Characterization and separation of semiconductor quantum dots and their conjugates by capillary electrophoresis.

Semiconductor quantum dots (QDs) are very important luminescent nanomaterials with a wide range of potential applications. Currently, QDs as labeling probes are broadly used in bioassays, including immunoassay, DNA hybridization, and bioimaging, due to their excellent physical and chemical properties, such as broad excitation spectra, narrow and size‐dependent emission profiles, long fluorescence life time, and good photostability. The characterization of QDs and their conjugates is crucial for their wide bioapplications. CE has become a powerful tool for the separation and characterization of QDs and their conjugates. In this review, some CE separation models of QDs are first introduced, mainly including CZE, CGE, MEKC, and ITP. And then, some key applications, such as the measurements of size, surface charge, and concentration of QDs and the characterization of QDs conjugates (e.g. QD–protein, QD–DNA, QD–small molecule), are also described. Finally, future perspectives are discussed.

Recent advances in chemiluminescence detection coupled with capillary electrophoresis and microchip capillary electrophoresis.

CE is an ideal analytical method for extremely volume‐limited biological microenvironments. However, the small injection volume makes it a challenge to achieve highly sensitive detection. Chemiluminescence (CL) detection is characterized by providing low background with excellent sensitivity because of requiring no light source. The coupling of CL with CE and MCE has become a powerful analytical method. So far, this method has been widely applied to chemical analysis, bioassay, drug analysis, and environment analysis. In this review, we first introduce some developments for CE–CL and MCE–CL systems, and then put the emphasis on the applications in the last 10 years. Finally, we discuss the future prospects.

Quantum dots trigger hot-start effects for pfu-based polymerase chain reaction

Hot-start (HS) effects were investigated in pfu-based polymerase chain reaction (PCR), when water-soluble CdTe quantum dots (QDs) were introduced in the PCR system. The HS effects were demonstrated by the higher amplicon yields and excellent suppression of non-specific amplification after pre-incubation of PCR mix with QDs between 35°C and 56°C. DNA targets were well amplified even after PCR mixture was pre-incubated 1 h at 50°C. Importantly, the effects of QDs nanoparticles could be reversed by increasing the pfu polymerase concentration, suggesting that there was an interaction between QDs and pfu DNA polymerase. Moreover, control experiment indicated that HS effect is not primarily due to the reduced pfu polymerase concentration resulted from the above interaction. Fluorescence correlation spectroscopy (FCS), a single molecule detection method, was used to investigate the possible mechanism of HS PCR with QDs. Preliminary FCS results suggested that CdTe QDs may directly interact with pfu DNA polymerase, rather than other components in the PCR system. Furthermore, results demonstrated that the interaction between QDs and pfu resulted in a reduction in pfu polymerase concentration. This study provided a good start to investigate potential implications of QDs in other key molecular biology techniques.

Size exclusion chromatography as a universal method for the purification of quantum dots bioconjugates

The bioconjugation of fluorescent quantum dots (QDs) and purification of QDs bioconjugates are of vital importance in bioapplications. In this paper, we systematically investigated the purification efficiency of QDs bioconjugates and their stability during separation process by using size exclusion chromatography (SEC) technique, fluorescence spectroscopy, and fluorescence correlation spectroscopy (FCS). In this study, commercial QDs and fluorescein isothiocyanate (FITC) were used as labeling probes, and bovine serum albumin (BSA) and antibody (Erbitux) were used as mode samples. The covalently linkage and the electrostatic interaction were used in bioconjugation procedures. We systematically studied the effects of certain factors such as the scales of column, loading volume, elution buffer, and separation media on the purification efficiency of QDs bioconjugates by a home‐built SEC device. And highly pure and monodisperse QDs bioconjugates could be obtained by SEC purification twice under the optimized conditions. Furthermore, we investigated the stability of QDs bioconjugates in different conjugation ways and purification conditions by FCS, and found that the stability of bioconjugates were poor when electrostatic binding mode was used. We also observed that Sephacryl S300 HR (separation range for globular proteins: 1 × 104–1.5 × 106 Da) was the best choice for purifying the vast majority of QDs‐bioconjugates. Our work described here will be constructive to popularization and applications of QDs in life science.

Homogeneous immunoassays by using photon burst counting technique of single gold nanoparticles.

In this paper, we reported a sensitive single particle method by combining the photon burst counting technique with gold nanoparticles (GNPs) as labeling probes. The photon bursting of single GNPs will be generated in a highly focused laser beam (less than 1 fL) due to the plasmon resonance scattering and Brownian motion of GNPs. We observed an excellent linear relationship between the photon burst counts and the number of particles in GNPs solution. We investigated the statistical behaviors of background noise and photon burst signal of GNPs, and proposed the data processing method based on Gaussian distribution of the background noise. A new homogeneous sandwich immunoassay was developed by using this single particle method. We evaluated the performance of this method by using prostate-specific antigen (PSA) as a model. The linear range of PSA was 1-1000 pmol/L and the detection limit was 0.8 pmol/L. This novel method was successfully used for the direct detection of cancer biomarker PSA in human serum samples. Our results were in good agreement with conventional ELISA assays.

A sensitive assay of mercury using fluorescence correlation spectroscopy of gold nanoparticles.

We described a new and sensitive method for the determination of mercury ions (Hg(2+) ) 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 Hg(2+) by two DNA thymine bases. When two GNPs labelled with different oligonucleotides were mixed with a sample containing Hg(2+), the T-Hg(2+)-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 Hg(2+). 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 Hg(2+). This new method was successfully applied for direct determination of Hg(2+) levels in water and cosmetics samples.