Yizhi Zhang

pH-sensitive nanocarrier based on gold/silver core-shell nanoparticles decorated multi-walled carbon manotubes for tracing drug release in living cells.

We fabricate a multifunctional nanocarrier based on multi-walled carbon nanotubes (MWCNTs) decorated with gold/silver core-shell nanoparticles (Au@Ag NPs) and fluorescein isothiocyanate (FITC) for tracking the intracellular drug release process. In the demonstrated nanocarrier, the Au@Ag NPs adsorbed on the surface of MWCNTs were labeled with the pH-dependent SERS reporter 4-Mercaptobenzoic acid (4MBA) for SERS based pH sensing. FITC was conjugated on MWCNTs to provide fluorescence signal for tracing the MWCNTs. Fluorescent doxorubicin (DOX) was used as the model drug which can be loaded onto MWCNTs via π-π stacking and released from the MWCNTs under acidic condition. By detecting the SERS spectrum of 4MBA, the pH value around the nanocarrier could be monitored. Besides, by tracing the fluorescence of FITC and DOX, we can also investigate the drug release process in cells. Experimental results show that the proposed nanocarrier retained a well pH-sensitive performance in living cells, and the DOX detached from MWCNTs inside the lysosomes and entered into the cytoplasm with the MWCNTs being left in lysosomes. To further investigate the drug release dynamics, 2-D color-gradient pH mapping were plotted, which were calculated from the SERS spectra of 4MBA. The detailed release process and carrier distribution have been recorded as environmental pH changes during cell endocytosis. Furthermore, we also confirmed that the proposed nanocarrier has a good biocompatibility. It indicates that the designed nanocarrier have a great potential in intraceable drug delivery, cancer cells imaging and pH monitoring.

Surface Enhanced Raman Scattering Based in Situ Hybridization Strategy for Telomere Length Assessment

Assessing telomere length is of vital importance since telomere length is closely related with several fatal diseases such as atherosclerosis and cancer. Here, we present a strategy to assess/measure telomere length, that is, surface enhanced Raman scattering (SERS) based in situ hybridization (SISH). The SISH method uses two kinds of SERS nanoprobes to hybridize in situ with telomeres and centromeres, respectively. The telomere specific SERS nanoprobe is called the Telo-probe, while the centromere specific SERS nanoprobe is called the Centro-probe. They are composed of metal nanoparticles (NPs), Raman reporter molecules and specially designed DNA strands. With longer telomeres, more Telo-probes will hybridize with them, resulting in a stronger SERS signal. To exclude possible influence of the SERS intensity by external factors (such as the nanoprobe concentration, the cell number or different batches of nanoprobes), centromeres are used as the inner control, which can be recognized by Centro-probes. Telomere length is evaluated using a redefined telomere-to-centromere ratio (T/C ratio). The calculation method for T/C ratio in SISH method is more reliable than that in fluorescent in situ hybridization (FISH). In addition, unlike FISH method, the SISH method is insensitive to autofluorescence. Moreover, SISH method can be used to analyze single telomeres. These features make SISH an excellent alternative strategy for telomere length measurement.

Facile detection of tumor-derived exosomes using magnetic nanobeads and SERS nanoprobes

Exosomes play an important role in intercellular communications. Here, we present a surface-enhanced Raman scattering (SERS) based detection method for tumor-derived exosomes using SERS nanoprobes and magnetic nanobeads. The SERS nanoprobe has a core–shell structure, with gold core–silver shell nanorods (Au@Ag NRs) as the SERS active core, Raman molecules as the SERS reporter and a silica layer as the protecting shell. The outmost surface of the SERS nanoprobe is decorated with exosome-specific antibodies. Each magnetic nanobead is fabricated by coating Fe3O4 nanoparticles (NPs) with a silica shell and attaching specific antibodies to the silica shell. With target exosomes present, the magnetic nanobeads and SERS nanoprobes can capture the exosomes by forming a sandwich-type immunocomplex. The immunocomplex (as well as the SERS nanoprobes) can be precipitated with a magnet, and thus SERS signals can be detected in the precipitates. With no target exosomes present, no immunocomplex can be formed, and thus quite weak SERS signals will be detected in the precipitates. Hence, the SERS signal of the final magnetic separation product can be used to detect exosomes. In the experiment, using one kind of tumor cells and one kind of normal cells, we proved that the presented method can be used for both qualitative and quantitative detection of tumor-derived exosomes.

Pharmacokinetics-on-a-Chip Using Label-Free SERS Technique for Programmable Dual-Drug Analysis

Synergistic effects of dual or multiple drugs have attracted great attention in medical fields, especially in cancer therapies. We provide a programmable microfluidic platform for pharmacokinetic detection of multiple drugs in multiple cells. The well-designed microfluidic platform includes two 2 × 3 microarrays of cell chambers, two gradient generators, and several pneumatic valves. Through the combined use of valves and gradient generators, each chamber can be controlled to infuse different kinds of living cells and drugs with specific concentrations as needed. In our experiments, 6-mercaptopurine (6MP) and methimazole (MMI) were chosen as two drug models and their pharmacokinetic parameters in different living cells were monitored through intracellular SERS spectra, which reflected the molecular structure of these drugs. The dynamic change of SERS fingerprints from 6MP and MMI molecules were recorded during drug metabolism in living cells. The results indicated that both 6MP and MMI molecules were diffused into the cells within 4 min and excreted out after 36 h. Moreover, the intracellular distribution of these drugs was monitored through SERS mapping. Thus, our microfluidic platform simultaneously accomplishes the functions to monitor pharmacokinetic action, distribution, and fingerprint of multiple drugs in multiple cells. Owing to its real-time, rapid-speed, high-precision, and programmable capability of multiple-drug and multicell analysis, such a microfluidic platform has great potential in drug design and development.

Dual peptides modified fluorescence-SERS dual mode imaging nanoprobes with improved cancer cell targeting efficiency

The field of cancer theragnostics has long been starving for imaging agents with an improved targeting efficiency. Herein, a dual receptor targeting nanoprobe with fluorescence-SERS dual mode imaging capacity has been demonstrated for the specific targeting of cervical cancer cells. First, silver nanoparticles were modified with Raman reporters and silica-protected fluorescence dyes to incorporate fluorescence-SERS dual mode imaging capacity. Then, two targeting peptides, transferrin receptor-specific peptide T7 and integrin ανβ3 bonding peptide RGD, were functionalized onto the outer surfaces of the nanoparticles, endowing the nanoprobes with enhanced recognition towards HeLa cells. Finally, to investigate the targeting capability of this synergetic probe, the cellular internalization was compared between single and dual targeting nanoprobes. The results from fluorescence-SERS dual-mode imaging demonstrated that the dual peptide functionalized nanoprobes not only promoted an elevated uptake by cancer cells, but were also responsible for an enhanced targeting ability towards cancer cells. This type of nanoprobe with dual-mode imaging and a dual-targeting ability would give a new prospect for the diagnosis and therapeutics of cancer.

In situ probing of cell–cell communications with surface-enhanced Raman scattering (SERS) nanoprobes and microfluidic networks for screening of immunotherapeutic drugs

Discovering novel drugs for cancer immunotherapy requires a robust in vitro drug screening platform that allows for straightforward probing of cell–cell communications. Here, we combined surface-enhanced Raman scattering (SERS) nanoprobes with microfluidic networks to monitor in situ the cancer–immune system intercellular communications. The microfluidic platform links up immune cells with cancer cells, where the cancer-cell secretions act as signaling mediators. First, gold@silver core–shell nanorods were employed to fabricate SERS immunoprobes for analysis of the signaling molecules. Multiple cancer secretions in a tumor microenvironment were quantitatively analyzed by a SERS-assisted three-dimensional (3D) barcode immunoassay with high sensitivity (1 ng/mL). Second, in an on-chip cell proliferation assay, multiple immunosuppressive proteins secreted by cancer cells were found to inhibit activation of immune cells, indicating that the platform simulates the physiological process of cancer–immune system communications. Furthermore, potential drug candidates were tested on this platform. A quantitative SERS immunoassay was performed to evaluate drug efficacy at regulating the secretion behavior of cancer cells and the activity of immune cells. This assay showed the suitability of this platform for in vitro drug screening. It is expected that the fully integrated and highly automated SERS-microfluidic platform will become a powerful analytical tool for probing intercellular communications and should accelerate the discovery and clinical validation of novel drugs.

Single molecule localization imaging of exosomes using blinking silicon quantum dots

Discovering new fluorophores, which are suitable for single molecule localization microscopy (SMLM) is important for promoting the applications of SMLM in biological or material sciences. Here, we found that silicon quantum dots (Si QDs) possess a fluorescence blinking behavior, making them an excellent candidate for SMLM. The Si QDs are fabricated using a facile microwave-assisted method. Blinking of Si QDs is confirmed by single particle fluorescence measurement and the spatial resolution achieved is about 30 nm. To explore the potential application of Si QDs as the nanoprobes for SMLM imaging, cell derived exosomes are chosen as the object owing to their small size (50-100 nm in diameter). Since CD63 is commonly presented on the membrane of exosomes, CD63 aptamers are attached to the surface of Si QDs to form nanoprobes which can specifically recognize exosomes. SMLM imaging shows that Si QDs based nanoprobes can indeed realize super resolved optical imaging of exosomes. More importantly, blinking of Si QDs is observed in water or PBS buffer with no need for special imaging buffers. Besides, considering that silicon is highly biocompatible, Si QDs should have minimal cytotoxicity. These features make Si QDs quite suitable for SMLM applications especially for live cell imaging.

Combining Multiplex SERS Nanovectors and Multivariate Analysis for In Situ Profiling of Circulating Tumor Cell Phenotype Using a Microfluidic Chip

Isolating and in situ profiling the heterogeneous molecular phenotype of circulating tumor cells are of great significance for clinical cancer diagnosis and personalized therapy. Herein, an on-chip strategy is proposed that combines size-based microfluidic cell isolation with multiple spectrally orthogonal surface-enhanced Raman spectroscopy (SERS) analysis for in situ profiling of cell membrane proteins and identification of cancer subpopulations. With the developed microfluidic chip, tumor cells are sieved from blood on the basis of size discrepancy. To enable multiplex phenotypic analysis, three kinds of spectrally orthogonal SERS aptamer nanovectors are designed, providing individual cells with composite spectral signatures in accordance with surface protein expression. Next, to statistically demultiplex the complex SERS signature and profile the cellular proteomic phenotype, a revised classic least square algorithm is employed to obtain the 3D phenotypic information at single-cell resolution. Combined with categorization algorithm partial least square discriminate analysis, cells from different human breast cancer subtypes can be reliably classified with high sensitivity and selectivity. The results demonstrate that this platform can identify cancer subtypes with the spectral information correlated to the clinically relevant surface receptors, which holds great potential for clinical cancer diagnosis and precision medicine.

Gold–carbon dots for the intracellular imaging of cancer-derived exosomes

As a novel fluorescent nanomaterial, gold-carbon quantum dots (GCDs) possess high biocompatibility and can be easily synthesized by a microwave-assisted method. Owing to their small sizes and unique optical properties, GCDs can be applied to imaging of biological targets, such as cells, exosomes and other organelles. In this study, GCDs were used for fluorescence imaging of exosomes. Tumor-specific antibodies are attached to the GCDs, forming exosome specific nanoprobes. The nanoprobes can label exosomes via immuno-reactions and thus facilitate fluorescent imaging of exosomes. When incubated with live cells, exosomes labeled with the nanoprobes can be taken up by the cells. The intracellular experiments confirmed that the majority of exosomes were endocytosed by cells and transported to lysosomes. The manner by which exosomes were taken up and the intracellular distribution of exosomes are unaffected by the GCDs. The experimental results successfully demonstrated that the presented nanoprobe can be used to study the intrinsic intracellular behavior of tumor derived exosomes. We believe that the GCDs based nanoprobe holds a great promise in the study of exosome related cellular events, such as cancer metastasis.

An innovative strategy to obtain extraordinary specificity in immunofluorescent labeling and optical super resolution imaging of microtubules

When performing immunofluorescent labeling of microtubules, Triton X-100 (TX100) is commonly used as the cell membrane permeabilization agent to improve the accessibility of antigens. Usually, before immunofluorescent labeling, cells are fixed first by aldehydes, followed by permeabilization with TX100. Here, we report an innovative immunofluorescent labeling strategy for microtubules with a meaningful alteration, that is, to treat cells with TX100 first and fix with aldehydes later. We proved that this subtle change can greatly improve the specificity of microtubular immunolabeling. However, treating cells first with TX100 can also severely disrupt the integrity of microtubules if an excessive amount of TX100 is used. Hence, TX100 is a “double-edged sword” in immunofluorescent labeling of microtubules and elaborative control of its dosage is required. In the experiment, we compared different immunofluorescent labeling protocols using various cell lines and found that treating cells with 0.02% TX100 before fixation is an optimal solution. Confocal laser scanning microscopy (CLSM), atomic force microscopy (AFM) and single molecule localization microscopy (SMLM) are utilized to verify the immunofluorescent labeling results performed via the presented unusual protocol. It is possible that such a modified immunofluorescent labeling protocol of microtubules can be generalized as a universal strategy.