Gufeng Wang

SERS as a bioassay platform: fundamentals, design, and applications

Bioanalytical science is experiencing a period of unprecedented growth. Drivers behind this growth include the need to detect markers central to human and veterinary diagnostics at ever-lower levels and greater speeds. A set of parallel arguments applies to pathogens with respect to bioterrorism prevention and food and water safety. This tutorial review outlines our recent explorations on the use of surface enhanced Raman scattering (SERS) for detection of proteins, viruses, and microorganisms in heterogeneous immunoassays. It will detail the design and fabrication of the assay platform, including the capture substrate and nanoparticle-based labels. The latter, which is the cornerstone of our strategy, relies on the construction of gold nanoparticles modified with both an intrinsically strong Raman scatterer and an antibody. This labelling motif, referred to as extrinsic Raman labels (ERLs), takes advantage of the well-established signal enhancement of scatterers when coated on nanometre-sized gold particles, whereas the antibody imparts antigenic specificity. We will also examine the role of plasmon coupling between the ERLs and capture substrate, and challenges related to particle stability, nonspecific adsorption, and assay speed.

Detection of the Potential Pancreatic Cancer Marker MUC4 in Serum Using Surface-Enhanced Raman Scattering

Pancreatic cancer (PC) is one of the most lethal malignancies. It has a 5-year survival rate of only 6%, owing in part to the lack of a reliable tumor marker for early diagnosis. Recent research has shown that the mucin protein MUC4 is aberrantly expressed in pancreatic adenocarcinoma cell lines and tissues but is undetectable in normal pancreas and chronic pancreatitis. Thus, the level of MUC4 in patient sera has the potential to function as a diagnostic and prognostic marker for PC. However, the measurement of MUC4 in sera using conventional test platforms (e.g., enzyme linked immunosorbent assay (ELISA) and radioimmunoassay (RIA)) has been unsuccessful. This has prevented the assessment of the utility of this protein as a possible PC marker in sera. In addressing this obstacle, the work herein examines the potential to create a simple diagnostic test for MUC4 through the development of a surface-enhanced Raman scattering (SERS)-based immunoassay, which was then used to demonstrate the first ever detection of MUC4 in cancer patient serum samples. Importantly, these measurements showed that sera from patients with PC produced a significantly higher SERS response for MUC4 compared to sera from healthy individuals and from patients with benign diseases. These results indicate that a SERS-based immunoassay can monitor MUC4 levels in patient sera, representing a much needed first step toward assessing the potential of this protein to serve as a serum marker for the early stage diagnosis of PC. This paper details these and other findings (i.e., the detection of the mucin protein CA19-9), which demonstrate that our SERS assay outperforms conventional assays (i.e., RIA and ELISA) with respect to limits of detection, readout time, and required sample volume.

Single Cell Optical Imaging and Spectroscopy

In his 1665 treatise, Micrographia, Robert Hooke described the many observations he had made using a microscope, including compartment-like structures in cork samples that he termed ‘cells’.1 In the three and a half centuries since Hooke’s day, both the microscope and our understanding of the cell have been vastly improved upon, and the current outlook suggests that the symbiotic relationship between the microscope and the cell will continue to flourish into the foreseeable future. The cell is a basic yet complicated ‘unit’ of interest to biology, just as the atom is to chemists. Ultimately, scientists want to ‘see to believe’ when it comes to an explanation of the complex inner workings of cells, but therein lies a complication. Seeing is not always a possibility in biological systems. Size, speed, sensitivity, and additional concerns plague the microscopist who wants to peek inside of a cell. Enter a variety of molecular and nanoparticle probes that are capable of tagging and pinpointing the location of biological components that would otherwise be invisible under the microscope. Advances in laser, camera, and imaging processing technologies have also played a crucial role in the burgeoning field of single cell imaging, because they have brought into view the fast processes that would normally escape the human eye. The purpose of this review is to highlight the key advances that have occurred in the past several years in the field of single cell optical imaging. It is not our intent to provide a comprehensive review of the types of experiments or the areas of cell research that are ongoing. Reviews with a distinctly biological flavor have been published recently, and these alternative reviews focus on specific details of the cell and the processes that occur within.2-7 Likewise, exceptional review papers that have discussed the full spectrum of nanoparticle probes and their properties have appeared recently.6-12 This review is designed to give an overview of the tools that are being specifically used to accomplish single cell imaging. As such, much of our emphasis in the first several sections of this paper is on imaging platforms, with a focus on design details that are important to single cell imaging experiments. Next we emphasize specific imaging experiments that highlight the types of findings that are possible at the nexus of microscopy, nanoprobes, and live cells. Particular attention is paid to the emerging orientation and rotational tracking of single probes linked to mechanistic functions and differentiated structures of biological interest. Finally, we provide a brief, yet rather complete, summary of single cell manipulation techniques.

Resolving rotational motions of nano-objects in engineered environments and live cells with gold nanorods and differential interference contrast microscopy.

Gold nanorods are excellent orientation probes due to their anisotropic optical properties. Their dynamic rotational motion in the 3D space can be disclosed with Nomarski-type differential interference contrast (DIC) microscopy. We demonstrate that by using the combination of gold nanorod probes and DIC microscopy, we are able to resolve rotational motions of nano-cargos transported by motor proteins at video rate not only on engineered surfaces but also on cytoskeleton tracks in live cells.

Mixed monolayers on gold nanoparticle labels for multiplexed surface-enhanced Raman scattering based immunoassays.

This paper describes a new approach, based on self-assembled mixed monolayers, to the design and preparation of extrinsic Raman labels (ERLs). ERLs function as spectroscopic tags for the readout of sandwich-type immunoassays using surface-enhanced Raman scattering (SERS). They are created by coating gold nanoparticles with Raman reporter molecules and antibodies specific for the target analyte. Mixed-monolayer ERLs are formed by covering gold nanoparticles with a mixture of two different thiolates. One thiolate serves to covalently bind antibodies to the particles, imparting biospecificity to the ERLs, while the other thiolate produces a strong Raman signal. Mixed-monolayer ERLs can be prepared in a few relatively simple steps using readily available materials. The SERS intensity of each type of ERL can be tuned to match other ERLs by adjusting the mixed monolayer composition, greatly facilitating the generation of sets of ERLs for multiplexed applications. The work herein not only describes the new pathway for ERL production, but also demonstrates the simultaneous qualitative and quantitative multiplexed detection using a set of four mixed-monolayer ERLs.

Single Particle Orientation and Rotation Tracking Discloses Distinctive Rotational Dynamics of Drug Delivery Vectors on Live Cell Membranes

Engineered nanoparticles have emerged as potentially revolutionary drug and gene delivery vectors. Using rod-shaped gold nanoparticles as a model, we studied for the first time the rotational dynamics of nanoparticle vectors on live cell membranes and its impact on the fate of these nanoparticle vectors. The rotational motions of gold nanorods with various surface modifiers were tracked continuously at 200 frames/s under a differential interference contrast microscope. We found that the rotational behaviors of gold nanorod vectors are strongly related to their surface charges. Specific surface functional groups and the availability of receptors on cell membranes also contribute to the rotational dynamics. The study of rotational brownian motion of nanoparticles on cell membranes will lead to a better understanding of the mechanisms of drug delivery and provide guidance in designing surface modification strategies for drug delivery vectors under various circumstances.

Wavelength-Dependent Differential Interference Contrast Microscopy: Selectively Imaging Nanoparticle Probes in Live Cells

Gold and silver nanoparticles display extraordinarily large apparent refractive indices near their plasmon resonance (PR) wavelengths. These nanoparticles show good contrast in a narrow spectral band but are poorly resolved at other wavelengths in differential interference contrast (DIC) microscopy. The wavelength dependence of DIC contrast of gold/silver nanoparticles is interpreted in terms of Mies theory and DIC working principles. We further exploit this wavelength dependence by modifying a DIC microscope to enable simultaneous imaging at two wavelengths. We demonstrate that gold/silver nanoparticles immobilized on the same glass slides through hybridization can be differentiated and imaged separately. High-contrast, video-rate images of living cells can be recorded both with and without illuminating the gold nanoparticle probes, providing definitive probe identification. Dual-wavelength DIC microscopy thus presents a new approach to the simultaneous detection of multiple probes of interest for high-speed live-cell imaging.

An exonuclease I-based label-free fluorometric aptasensor for adenosine triphosphate (ATP) detection with a wide concentration range

A novel aptamer-based label-free assay for sensitive and selective detection of ATP was developed. This assay employs a new aptamer/fluorescent probe system that shows resistance to exonuclease I (Exo I) digestion upon binding to ATP molecules. In the absence of ATP, the complex between the ATP-binding aptamer (ATP-aptamer) and a DNA binding dye, berberine, is digested upon the addition of exonuclease I, leading to the release of berberine into solution and consequently, quenched berberine fluorescence. In the presence of ATP, the ATP-binding aptamer folds into a G-quadruplex structure that is resistant to Exo I digestion. Accordingly, berberine is protected in the G-quadruplex structure and high fluorescence intensity is observed. As such, based on the fluorescence signal change, a label-free fluorescence assay for ATP was developed. Factors affecting the analysis of ATP including the concentration of ATP-binding aptamer, reaction time, temperature and the concentration of Exo I were comprehensively investigated. Under optimal conditions, the fluorescence intensity of the sensing system displayed a response for ATP in a wide range up to 17.5 mM with a detection limit of 140 nM.

FORCE GENERATION AND PHOSPHATE RELEASE STEPS IN SKINNED RABBIT SOLEUS SLOW-TWITCH MUSCLE FIBERS

The force-generation and phosphate-release steps of the cross-bridge cycle in rabbit soleus slow-twitch muscle fibers (STF) were investigated using sinusoidal analysis, and the results were compared with those of rabbit psoas fast-twitch fibers (FTF). Single fiber preparations were activated at pCa 4.40 and ionic strength 180 mM at 20 degrees C. The effects of inorganic phosphate (Pi) concentrations on three exponential processes, B, C, and D, were studied. Results are consistent with the following cross-bridge scheme: [formula: see text] where A is actin, M is myosin, D is MgADP, and P is inorganic phosphate. The values determined are k4 = 5.7 +/- 0.5 s-1 (rate constant of isomerization step, N = 9, mean +/- SE), k-4 = 4.5 +/- 0.5 s-1 (rate constant of reverse isomerization), K4 = 1.37 +/- 0.13 (equilibrium constant of the isomerization), and K5 = 0.18 +/- 0.01 mM-1 (Pi association constant). The isomerization step (k4) in soleus STF is 20 times slower, and its reversal (k-4) is 20 times slower than psoas fibers. Consequently, the equilibrium constant of the isomerization step (K4) is the same in these two types of fibers. The Pi association constant (K5) is slightly higher in STF than in FTF, indicating that Pi binds to cross-bridges slightly more tightly in STF than FTF. By correlating the cross-bridge distribution with isometric tension, it was confirmed that force is generated during the isomerization (step 4) of the AMDP state and before Pi release in soleus STF.

Three-Dimensional Super-Localization and Tracking of Single Gold Nanoparticles in Cells

We introduce a precise three-dimensional (3D) localization method of spherical gold nanoparticle probes using model-based correlation coefficient mapping. To accomplish this, a stack of sample images at different z-positions are acquired, and a 3D intensity profile of the probe serving as the model is used to map out the positions of nanoparticles in the sample. By using this model-based correlation imaging method, precise localization can be achieved in imaging techniques with complicated point spread functions (PSF) such as differential interference contrast (DIC) microscopy. We demonstrated the localization precision of 4-7 nm laterally and 16 nm axially for 40-nm gold nanospheres at an imaging rate of 10 frames per second. The 3D superlocalization method was applied to tracking gold nanospheres during live endocytosis events.