Yingyue Zhu

Side‐by‐Side and End‐to‐End Gold Nanorod Assemblies for Environmental Toxin Sensing

Controllable assembly of nanoscale building blocks (monomers) is a necessary part of practical realization of the unique optical, electrical, magnetic, and chemical properties of nanoscale matter in macroscale materials. Such assemblies also contain much fundamental information about collective behavior of nanocolloids, which we are just beginning to understand. The key decisive factors for the successful assembly of nanocolloids is the anisotropy of nanoscale interactions, which stems from both the shape of nanocolloids and unequal distribution of organic molecules on their surface. Gold nanorods (Au NRs) have both geometrical and chemical anisotropy components and demonstrate strong optical extinction in the range of visible and near-infrared (NIR) wavelengths convenient for both research and practical purposes. Au NRs can be assembled by interactions with organic molecules, polymers, an antibody–antigen reaction, biotin–strepavidin connectors, and DNA, leading to superstructures with different degree of organization and complexity of collective behavior. Besides the utilization of NR monomers in non-linear optics, cellular imaging, and cancer therapy, optical effects corresponding to monomer!superstructure transitions allowed preparation of excellent biosensors because of large changes in oscillation frequencies of plasmons when NR pairs are formed. These studies mostly targeted biomedical applications. Simultaneously, their unique sensing capabilities have been virtually unexplored for the needs of environmental detection and monitoring. These challenges and impact can equal or exceed those encountered in detection of cancer. A better understanding of methods for the realization of speed/selectivity/sensitivity detection of common environmental pollutants is thus of great importance. Therefore, we decided to explore the potential of NR assemblies taking a pervasive environmental toxin, namely microcystin-LR (MC-LR), as the model while also addressing the general questions about the choice of different assembly motif for different sensing tasks. MC-LR is common in both developed and developing countries, with recorded cases of mass poisoning. MC-LR originates from common bluegreen algae and causes rapid liver failure; prolonged exposure to small concentrations of MC-LR in drinking water causes liver cancer. Herein, we describe the successful use of Au NRs for detection of MC-LR, which is significantly more sensitive than the traditional techniques, such as ELISA, yielding detection limit of 5 pgmL . It is also much simpler and faster than any other methods. These two factors are critical for environmental monitoring and have been a long-standing challenge. The pattern of the assembly strongly affects the sensitivity parameters for MC-LR detection. To realize different modes of assembly, such as side-byside and end-to-end motifs, with a degree of control sufficient for conclusive evaluation of sensing implications, two kinds of protein-carrying Au NRs were synthesized (Figure 1). One type of NR carried MC-LR antibodies (ABs) preferentially on the sides, while the other type carried antibodies located almost exclusively in the ends. These motifs were formed by using either electrostatic binding or covalent attachment of the antibodies mediated by a bifinctional linker, thioctic acid (TA).When electrostatic forces govern the placement of ABs, they attach primarily to the sides of NRs due to the larger area of contact and thus stronger electrostatic interactions. When a TA anchor covalently binds by a S Au bond, the conjugation of the ABs occurs predominantly in the ends of the rods due to better accessibility of the gold surface to the reactive thiol end. Variation of pH also allows varying repulsion or attraction of NRs and MC-LR, modality of attachment, and geometrical characteristics of assemblies (see the Supporting Information). The number of AB molecules on the surface of one Au NR was estimated to be 31 and 10 for the side-by-side [*] L. Wang, Y. Zhu, L. Xu, Dr. W. Chen, H. Kuang, L. Liu, Prof. C. Xu School of Food Science and Technology, State Key Laboratory of Food Science and Technology Jiangnan University, Wuxi, 214122 (China) E-mail: xcl@jiangnan.edu.cn Dr. W. Chen, A. Agarwal, Prof. N. A. Kotov Department of Chemical Engineering, Department of Biomedical Engineering Department of Materials Science and Engineering University of Michigan, Ann Arbor, MI 48109 (USA) E-mail: kotov@umich.edu [] These authors contributed equally to this paper.

Fabricated aptamer-based electrochemical "signal-off" sensor of ochratoxin A.

An ultrasensitive and rapid electrochemical platform for the specific detection of ochratoxin A (OTA) was developed. In this method, three single-stranded DNA molecules, including the aptamer, were immobilized on the surface of an electrode. Binding of the OTA target analyte to the aptamer changed the redox current of methylene blue (MB), which was used as the electrochemical probe, in a manner that was dependent on OTA concentration. With signal enhancement from gold nanoparticle-functionalized DNA, the sensitivity of this method for OTA was as low as 30 pg/mL, and the effective sensing range was from 0.1 to 20 ng/mL. To investigate the sensing process, the conformational switch of the aptamer was studied by circular dichroism (CD), which confirmed the recognition of the aptamer by the target OTA. Given its sensitivity and rapid detection, we believe this approach has the potential to be a main technology for the detection of toxins in the field of food safety, and in other areas.

Simple, rapid, sensitive, and versatile SWNT-paper sensor for environmental toxin detection competitive with ELISA

Safety of water was for a long time and still is one of the most pressing needs for many countries and different communities. Despite the fact that there are potentially many methods to evaluate water safety, finding a simple, rapid, versatile, and inexpensive method for detection of toxins in everyday items is still a great challenge. In this study, we extend the concept of composites, impregnated porous fibrous materials, such as fabrics and papers, by single-walled carbon nanotubes (SWNTs), toward very simple but high-performance biosensors. They utilize the strong dependence of electrical conductivity through nanotubes percolation network on the width of nanotube-nanotube tunneling gap and can potentially satisfy all the requirements outlined above for the routine toxin monitoring. An antibody to the microcystin-LR (MC-LR), one of the common culprits in mass poisonings, was dispersed together with SWNTs. This dispersion was used to dip-coat the paper rendering it conductive. The change in conductivity of the paper was used to sense the MC-LR in the water rapidly and accurately. The method has the linear detection range up to 10 nmol/L and nonlinear detection up to 40 nmol/L. The limit of detection was found to be 0.6 nmol/L (0.6 ng/mL), which satisfies the strictest World Health Organization standard for MC-LR content in drinking water (1 ng/mL) and is comparable to the detection limit of the traditional ELISA method of MC-LR detection, while drastically reducing the time of analysis by more than an order of magnitude, which is one of the major hurdles in practical applications. Similar technology of sensor preparation can also be used for a variety of other rapid environmental sensors.

An aptamer-based chromatographic strip assay for sensitive toxin semi-quantitative detection

An aptamer-based chromatographic strip assay method for rapid toxin detection was developed. The aptamer-based strip assay was based on the competition for the aptamer between ochratoxin A and DNA probes. The sensing results indicated that the sensitivity of the aptamer-based strip was better than that of conventional antibody-based strips. The visual limit of detection of the strip for qualitative detection was 1 ng/mL while the LOD for semi-quantitative detection could down to 0.18 ng/mL by using scanning reader. The recoveries of test samples were from 96% to 110%. All detections could be achieved in less than 10 min, indicating that the aptamer-based strip could be a potential useful tool for rapid on-site detections.

Crown ether assembly of gold nanoparticles: Melamine sensor

Melamine toxicity causing the renal failure and death of animals and humans has recently attracted worldwide attention. Developing an easy, fast, and sensitive method for the routine melamine detection is of great importance. Herein, we report the colorimetric sensing of melamine, based on the 18-crown-6 ether functionalized gold nanoparticles (GNPs) through the formation of cavity complexes with amines. Based on high extinction coefficients and spectral sensitivity of the surface plasmon resonance band of the GNPs, the rapid and sensitive melamine detection was achieved both visually and spectroscopically. Under the optimal conditions, melamine could be selectively detected in a concentration range from 10 to 500 ppb with a limit of detection as 6 ppb (3σ), which is much lower than the strictest melamine safety requirement of 1 ppm. To demonstrate the selectivity and practicality of the method, melamine detection was realized in the real complex samples (dairy) with excellent analyte concentration recovery, indicating its applicability for real-time monitoring of toxins in common products. Crown ether assembly of GNP also opens a new route for the formation of three-dimensional pseudorotaxane-like assemblies of nanoparticles that can be applicable to a variety of amine-bearing ligands.

Gold nanorod assembly based approach to toxin detection by SERS

A novel, rapid and ultrasensitive surface-enhanced Raman scattering (SERS) immunoassay for the detection of microcystin LR was developed based on the assembly of gold nanorods (GNRs). GNRs were assembled into nanorod chains through the bio-recognition. The SERS signal of the probe molecule modified to the end of the NRs could be enhanced due to the hot spot between the NRs. Meanwhile, the finite integration technique simulation revealed that the electrical field of nanorods was increased obviously depending on the degree of end to end assembled structures. The research results demonstrated that the linear detection range was from 0.01 ng mL−1 to 5 ng mL−1, the limit of detection was 5 pg mL−1, and the time necessary for the analysis was only 15 min.

Effects of Quantum Dots in Polymerase Chain Reaction

The effects of quantum dots (QDs) on the elimination of nonspecific amplification of the polymerase chain reaction (PCR) were investigated. It was found that QDs could increase the specificity of the PCR at different annealing temperatures and with DNA templates of different lengths. The effects of QDs on the efficiency of the PCR were also studied, and the results showed that there was no enhancement. The mechanisms underlying these effects are discussed. This method could be used to modify the amplification results of the conventional PCR. Furthermore, this technology could make the PCR more widely applicable, especially in the multi-PCR reaction system with different annealing temperatures. This is of great significance for gene diagnosis.

Ultrasensitive Detection of Trace Protein by Western Blot Based on POLY-Quantum Dot Probes

In this study, we describe an ultrasensitive quantum dots (QDs)-based Western blot. With the high affinity of avidin-functionalized POLY-QDs and simplification of the detection process, this enabled the quantitative analysis of protein by Western blotting. To prepare the POLY-QDs, CdTe quantum dots were first coated with biotinylated denatured bovine serum albumin and then, via the effect of the biotin-avidin system, the biotinylated denatured bovine serum albumin-coated QDs, which had strong fluorescence, were linked together. With this series of modifications, the fluorescence intensity of CdTe QDs was significantly increased. Using the POLY-QDs as labels, the signal of Western blotting was more sensitive in tracing the protein than traditional dyeing. In the present study, trace protein A was applied to POLY-QDs-based Western blotting as a model. The linearity of this method was from 30 pg to 1.5 ng, and the sensitivity was up to low pictogram values. The final fluorescence signal on the polyvinylidenedifluoride (PVDF) membrane was retained for at least 40 min. The results of this study indicate that the POLY-QDs-based Western blot is an excellent quantitative analytical method for trace protein analysis.

Simple, rapid and sensitive detection of antibiotics based on the side-by-side assembly of gold nanorod probes

In this work, we report on the application of versatile gold nanorods (GNRs) in optical sensors for the detection of antibiotics. The target analyte, Gentamicin (GM) and ovalbumin (OVA)-antigen-modified GNRs together competed with antibody-modified GNRs, then influenced the formation of side-by-side aggregates of the GNRs by antibody-antigen interactions. Accordingly, the UV-vis absorption intensity of the side-by-side aggregates was changed in the presence of the target analyte. This assay allowed the selective determination of GM in the range of 0.1-20 ng/mL, and the limit of detection (LOD) of GM was 0.05 ng/mL. Furthermore, compared with the traditional plate-based immunoassay, the developed method was easy to perform without washing cycles and the results could be read as soon as the nanoprobe-analyte incubation was complete. Therefore, the developed method could be a promising tool for the detection of antibiotic residues.

G-quadruplex DNAzyme-based microcystin-LR (toxin) determination by a novel immunosensor.

In this paper, we demonstrate the application of versatile G-quadruplex-hemin DNAzymes in an immunoassay for detecting Microcystin-LR (MC-LR). Taking advantage of the high peroxidase activity of G-quadruplex-hemin complexes and the enhancement effect of gold nanoparticles (AuNPs), the method showed simple, high sensitive and selectivity detection of target toxin residues in water samples. The coated antigen, MC-LR-ovalbumin (OVA) coated on a plate, competed for MC-LR antibody with added target analyte to form antibody-antigen immune complexes. Subsequently, the immune complex reacted with G-quadruplex-labeled secondary antibodies for colorimetric detection of MC-LR. This assay specifically determined MC-LR in the linear range of 0.1-10 ng/ml, with a limit of detection (LOD) of 0.05 ng/mL for MC-LR. The results indicated that the novel immunoassay was an alternative to traditional plate-based immunoassay for MC-LR residue screening due to this method met the standard of World Health Organization (WHO) requirements for MC-LR content in drinking water (1 ng/mL).