Single Living Cell Analysis Decodes Dynamical Signaling Patterns Triggering Different Phenotypes of Cell Migration.

A sound understanding of cell migration behaviors and the internal mechanism is crucial for studying cancer metastasis and invasion. Continuous cell tracking and quantifying cellular and molecular dynamics of cell migration at the single-cell level is essential to elucidate rare, dynamic, and heterogeneous cell responses. Yet, a competent comprehensive analytical platform is lacking. Herein, we present an integrated single living cell analysis platform that enables long-term observation of migration behavioral phenotypes in single cells and simultaneous analysis of the signaling proteins and complexes during cell migration. Considering correlation between pathways and phenotypes, this platform is capable of analyzing multiple phenotypes and signaling protein dynamics at a subcellular resolution, reflecting the molecular mechanism of biological behavior. Using the EGFR-PI3K signaling pathway as a proof-of-principle, we explored how the pathway and related regulators, Rho GTPases, promote different migration phenotypes. Signaling pathway protein complexes p85α-p110α and p85α-PTEN were found to reciprocally modulate each other and subsequently regulate the expression level of the small GTPases by EGFR-related signaling pathways, which governs the cell migratory behavior. Thus, this single-cell analysis platform is a promising tool for rapid molecular mechanism analysis and direct observation of migration phenotypes at the single-cell level, providing insights into the molecular mechanism and phenotypes in cell migration.

Single-Cell Plasmonic Immunosandwich Assay Reveals the Modulation of Nucleocytoplasmic Localization Fluctuation of ABL1 on Cell Migration.

Cell migration is an essential process of cancer metastasis. The spatiotemporal dynamics of signaling molecules influences cellular phenotypic outcomes. It has been increasingly documented that the Abelson (ABL) family kinases play critical roles in solid tumors. However, ABL1's shuttling dynamics in cell migration still remains unexplored. This is mainly because tools permitting the investigation of translocation dynamics of proteins in single living cells are lacking. Herein, to bridge this gap, we developed a unique multifunctional integrated single-cell analysis method that enables long-term observation of cell migration behavior and monitoring of signaling proteins and complexes at the subcellular level. We found that the shuttling of ABL1's to the cytoplasm results in a higher migration speed, while its trafficking back to the nucleus leads to a lower one. Furthermore, our results indicated that fluctuant protein-protein interactions between 14-3-3 and ABL1 modulate ABL1's nucleocytoplasmic fluctuation and eventually affect the cell speed. Importantly, based on these new insights, we demonstrated that disturbing ABL1's nuclear export traffic and 14-3-3-ABL1 complexes formation can effectively suppress cell migration. Thus, our method opens up a new possibility for simultaneous tracking of internal molecular mechanisms and cell behavior, providing a promising tool for the in-depth study of cancer.

Probing Protein 4'-Phosphopantetheinylation in Single Living Cells.

4'-Phosphopantetheinylation (4PPTylation) of proteins, which is derived from the hydrolysis of coenzyme A (CoA), is an essential post-translational modification participating in biosynthetic and metabolic pathways. However, due to the lack of specific recognition ligands as well as the shortage of sensitive analytical tools for single-cell analysis, the in-depth exploration of new cellular functions and mechanisms of protein 4PPTylation has been much hampered. In this study, we rationally engineered CoA-imprinted Raman nanotags for the specific recognition of 4PPTylation and thereby developed a molecularly imprinted polymer (MIP)-based plasmonic immunosandwich assay (PISA) for facile probing the 4PPTylation of ALDH1L1 in single cells. The molecularly imprinted nanotags exhibited excellent binding properties, giving a dissociation constant of 10-6 M and cross-reactivity values of less than 10%. The MIP-based PISA enabled the specific and sensitive detection of the level of 4PPTylated ALDH1L1 in single living cells. Particularly, monitoring of the fluctuation of 4PPTylated ALDH1L1 in single cells under simulation by an inhibitor (methotrexate) that acts on a different metabolism pathway was achieved, implying possible crosstalk between two different pathways in folate metabolism. Thus, the imprinted Raman nanotags-PISA provides a promising analytical tool with a single-cell resolution for exploring new functions and elucidating their mechanisms of protein 4PPTylation.

Probing Queuosine Modifications of Transfer RNA in Single Living Cells via Plasmonic Affinity Sandwich Assay.

Queuosine (Q) modification on tRNA plays an essential role in protein synthesis, participating in many tRNA functions such as folding, stability, and decoding. Appropriate analytical tools for the measurement of tRNA Q modifications are essential for the exploration of new roles of Q-modified tRNAs and the rationalization of their exact mechanisms. However, conventional methods for Q modification analysis suffer from apparent disadvantages, such as destructive cells, tedious procedure, and low sensitivity, which much hamper in-depth studies of Q modification-related biological questions. In this study, we developed a new approach called plasmonic affinity sandwich assay that allows for facile and sensitive determination of Q-modified tRNAs in single living cells. This method relies on the combination of plasmon-enhanced Raman scattering detection, base-paring affinity in-cell microextraction, and a set of boronate affinity and molecularly imprinted labeling nanotags for selective recognition of individual Q modifications, including queuosine, galactosyl queuosine (Gal-Q), and mannosyl queuosine (Man-Q). The developed method exhibited high affinity extraction and high specificity recognition. It allowed for the measurement of tRNA Q modifications in not only Q-rich cultured tumor cells but also Q-deficient primary tumor cells. Usefulness of this approach for investigation of the change of the Q modification level in single cells under oxidative stress was demonstrated. Because of its significant advantages over conventional methods, this approach provides a promising analytical tool for the exploration of more roles of Q-modified tRNAs and elucidation of their mechanisms.

Multiplexed Single-Cell Plasmonic Immunoassay of Intracellular Signaling Proteins Enables Non-Destructive Monitoring of Cell Fate.

It is of significant importance in cancer biology to identify signaling pathways that play key roles in cell fate determination. Dissecting cellular signaling pathways requires the measurement of a large number of signaling proteins. However, tools for simultaneously monitoring multiple signaling pathway components in single living cells remain limited at present. Herein, we describe an approach, termed multiplexed single-cell plasmonic immunosandwich assay (mxscPISA), for simultaneous detection of multiple signaling proteins in individual living cells. This approach enabled simultaneous non-destructive monitoring of multiple (up to five, currently the highest multiplexing capacity in living cells) cytoplasmic and nucleus signaling proteins in individual cells with ultrahigh detection sensitivity. As a proof of principle, the epidermal growth factor receptor (EGFR) pathway, which plays a central role in cell fate determination, was investigated using this approach in this study. We found that there were differential attenuation rate of pro-survival and accumulation rate of pro-death signaling protein of the EGFR pathway in response to EGFR inactivation. These findings implicate that, after EGFR inactivation, a transient imbalance between survival and apoptotic signaling outputs contributed to the final cell fate of death. The mxscPISA approach can be a promising tool to reveal a signaling dynamic pattern at the single-cell level and to identify key components of signaling pathways that contribute to the final cell fate using only a limited number of cells.

Single-Cell Analysis of Signaling Proteins Provides Insights into Proapoptotic Properties of Anticancer Drugs.

Single-cell DNA analysis technology has provided unprecedented insights into many physiological and pathological processes. In contrast, technologies that allow protein analysis in single cells have lagged behind. Herein, a method called single-cell Plasmonic ImmunoSandwich Assay (scPISA) that is capable of measuring signaling proteins and protein complexes in single living cells is described. scPISA is straightforward, comprising specific in-cell extraction and ultrasensitive plasmonic detection. It is applied to evaluate the efficacy and kinetics of cytotoxic drugs. It reveals that different drugs exhibit distinct proapoptotic properties at the single-cell level. A set of new parameters is thus proposed for comprehensive and quantitative evaluation of the efficacy of anticancer drugs. It discloses that metformin can dramatically enhance the overall anticancer efficacy when combined with actinomycin D, although it itself is significantly less effective. Furthermore, scPISA reveals that survivin interacts with cytochrome C and caspase-3 in a dynamic fashion in single cells during continuous drug treatment. As compared with conventional assays, scPISA exhibits several significant advantages, such as ultrahigh sensitivity, single-cell resolution, fast speed, and so on. Therefore, this approach may provide a powerful tool for wide, important applications from basic research to clinical applications, particularly precision medicine.

Efficient Mass Spectrometric Dissection of Glycans via Gold Nanoparticle-Assisted in-Source Cation Adduction Dissociation.

Structural identification of glycans is important but remains challenging, for which tandem mass spectrometry has evolved as an indispensable tool. However, it requires additional complex hardware and extra time for ion extraction. Herein, we report a straightforward approach called gold nanoparticles (AuNPs)-assisted in-source cation adduction dissociation (isCAD) for efficient mass spectrometry (MS) dissection of glycans. Although AuNPs have been employed as an inorganic matrix for MALDI MS, this is the first report of AuNP-induced fragmentation. In this approach, AuNPs were employed as an energy absorber for laser ionization as well as a trigger for fragmentation, while residual or deliberately added sodium ions acted as a cationizing agent. The addition of sodium ions induced intensive fragmentation, but the addition of protons suppressed the fragmentation, allowing for facile tuning of the degree of fragmentation. In addition, it was found that larger oligosaccharides and glycans were much easier to fragment as compared with their smaller counterparts, and the use of high-concentration AuNPs effectively suppressed the degree of fragmentation and thereby provided abundant molecular ions. Without any extra hardware and ion extraction, this approach provides a straightforward, cost-efficient and tunable fragmentation for efficient MS dissection of saccharides, including monosaccharides, oligosaccharides, and glycans. Thus, it opens new access to efficient MS dissection of glycans.

Dual Molecularly Imprinted Polymer-Based Plasmonic Immunosandwich Assay for the Specific and Sensitive Detection of Protein Biomarkers.

Molecularly imprinted polymers (MIPs), which are synthesized in the presence of a template, have been widely used as antibody mimics for important applications. Through the combination with a highly sensitive detection scheme such as chemiluminescence and surface-enhanced Raman scattering (SERS), MIP-based sandwich assays have emerged as promising analytical tools for the detection of disease biomarkers. However, so far, MIPs have been used only as target-capturing probes, whereas labeling by other means was needed, which limits the application range. Herein, we present a new approach, called a dual MIP-based plasmonic immunosandwich assay (duMIP-PISA), for the specific and sensitive detection of protein biomarkers in complex biological samples. A C-terminal epitope-imprinted self-assembled gold nanoparticle monolayer-coated glass slide was prepared as a plasmonic substrate for the specific extraction of target protein, while N-terminal epitope-imprinted Raman-responsive Ag@SiO2 nanoparticles were prepared as nanotags for the specific labeling of captured protein. The formed MIP-protein-MIP sandwich-like complexes could produce a significantly enhanced SERS signal. The dual MIP-based recognitions ensured high specificity of the assay, while SERS detection provided ultrahigh sensitivity. The duMIP-PISA of neuron-specific enolase (NSE) in human serums was demonstrated, which permitted the differentiation of small cell lung cancer patients from healthy individuals. As compared to regular ELISA, the duMIP-PISA exhibited multiple merits including a simpler procedure, faster speed, lower sample volume requirement, and wider linear range. The approach well demonstrated the great potentials of MIPs and can be easily modified and extended to other protein biomarkers. Therefore, the duMIP-PISA approach holds great promise in many important applications such as disease diagnosis.

Pattern Recognition of Cells via Multiplexed Imaging with Monosaccharide-Imprinted Quantum Dots.

Recognition of cancer cells is essential for many important areas such as targeted cancer therapy. Multimonosaccharide-based recognition could be a useful strategy to improve the recognition specificity, but such a possibility has not been explored yet. Herein we report pattern recognition of cells via multiplexed imaging with monosaccharide-imprinted quantum dots (QDs). Imprinted with sialic acid, fucose, and mannose as the template, respectively, the QDs exhibited good specificity toward the template monosaccharides. Multiplexed imaging of cells simultaneously stained with these monosaccharide-imprinted QDs revealed the relative expression levels of the monosaccharides on the cells. Pattern recognition constructed using the intensities of multiplexed imaging unveiled the similarities and differences of different cell lines, allowing for the recognition of not only cancer cells from normal cells but also cancer cells of different cell lines. Thus, this study paved a solid ground for the design and preparation of novel cancer-cell targeting reagents and nanoprobes.

N-glycan biosignatures as a potential diagnostic biomarker for early-stage pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis, with a 5-year survival rate of less than 10%, owing to its late-stage diagnosis. Early detection of pancreatic cancer (PC) can significantly increase survival rates. AIM: To identify the serum biomarker signatures associated with early-stage PDAC by serum N-glycan analysis. METHODS: An extensive patient cohort was used to determine a biomarker signature, including patients with PDAC that was well-defined at an early stage (stages I and II). The biomarker signature was derived from a case-control study using a case-cohort design consisting of 29 patients with stage I, 22 with stage II, 4 with stage III, 16 with stage IV PDAC, and 88 controls. We used multiparametric analysis to identify early-stage PDAC N-glycan signatures and developed an N-glycan signature-based diagnosis model called the "Glyco-model". RESULTS: The biomarker signature was created to discriminate samples derived from patients with PC from those of controls, with a receiver operating characteristic area under the curve of 0.86. In addition, the biomarker signature combined with cancer antigen 19-9 could discriminate patients with PDAC from controls, with a receiver operating characteristic area under the curve of 0.919. Glyco-model demonstrated favorable diagnostic performance in all stages of PC. The diagnostic sensitivity for stage I PDAC was 89.66%. CONCLUSION: In a prospective validation study, this serum biomarker signature may offer a viable method for detecting early-stage PDAC.

Probing low-copy-number proteins in single living cells using single-cell plasmonic immunosandwich assays.

Cellular heterogeneity is pervasive and of paramount importance in biology. Single-cell analysis techniques are indispensable for understanding the heterogeneity and functions of cells. Low-copy-number proteins (fewer than 1,000 molecules per cell) perform multiple crucial functions such as gene expression, cellular metabolism and cell signaling. The expression level of low-copy-number proteins of individual cells provides key information for the in-depth understanding of biological processes and diseases. However, the quantitative analysis of low-copy-number proteins in a single cell still remains challenging. To overcome this, we developed an approach called single-cell plasmonic immunosandwich assay (scPISA) for the quantitative measurement of low-copy-number proteins in single living cells. scPISA combines in vivo microextraction for specific enrichment of target proteins from cells and a state-of-the-art technique called plasmon-enhanced Raman scattering for ultrasensitive detection of low-copy-number proteins. Plasmon-enhanced Raman scattering detection relies on the plasmonic coupling effect (hot-spot) between silver-based plasmonic nanotags and a gold-based extraction microprobe, which dramatically enhances the signal intensity of the surface-enhanced Raman scattering of the nanotags and thereby enables sensitivity at the single-molecule level. scPISA is a straightforward and minimally invasive technique, taking only ~6-15 min (from in vivo extraction to Raman spectrum readout). It is generally applicable to all freely floating intracellular proteins provided that appropriate antibodies or alternatives (for example, molecularly imprinted polymers or aptamers) are available. The entire protocol takes ~4-7 d to complete, including material fabrication, single-cell manipulation, protein labeling, signal acquisition and data analysis.

Specific recognition of proteins and peptides via controllable oriented surface imprinting of boronate affinity-anchored epitopes.

Molecularly imprinted polymers (MIPs) are chemically synthesized materials mimicking the recognition of antibodies towards antigens. Epitope imprinting has been an effective strategy, making imprinting of proteins flexible to a great extent. However, so far there is apparently a lack of facile and versatile epitope imprinting approaches. Herein, we present a new method called controllable oriented surface imprinting of boronate affinity-anchored epitopes. In this method, a C-terminus nonapeptide epitope was glycated and anchored as a template onto a boronic acid-functionalized substrate, followed by controllable oriented surface imprinting via the polycondensation of multiple silylating reagents containing functionalities capable of interacting with the epitope. The developed imprinting approach allowed for precise control of the thickness of the imprinting layer through adjusting the imprinting time, generating excellent binding properties. This method was verified to be versatile and efficient. Thus, it could greatly facilitate the preparation of MIPs for specific recognition of proteins and peptides.

Recent advances in nanostructure/nanomaterial-assisted laser desorption/ionization mass spectrometry of low molecular mass compounds.

Laser desorption ionization mass spectrometry (LDI-MS) has been developed to be an essential tool for the analysis and identification of a large variety of species, ranging from small metabolites, drugs and pollutants to peptides and proteins and even to bacteria. Matrix assisted LDI-MS has shown a great power for macromolecules, but its analytical capability to low mass compounds is limited due to inherent drawbacks, including abundant interference in low mass range (<800 Da) caused by matrix self-dissociation and poor reproducibility due to inhomogeneous crystallization. Several matrix-free methods have been developed to solve these issues, mainly through altering organic matrix with inorganic nanostructured surfaces or nanomaterials as LDI medium. In the past five years, improvements on conventional silicon-based, metal-based, metal oxide-based and carbon-based LDI media as well as emerging novel materials such as 2D nanomaterials and organic frameworks have gained important progresses. Meanwhile, with the deep research in light-matter interaction, LDI mechanism studies begin to grow and become to attach great importance. Advances in both medium and mechanism promote to expand the applicable potential of nanostructure/nanomaterial-assisted LDI-MS in many aspects, including trace analysis, structural analysis and MS imaging. In this review, we survey the major progresses in this area in recent years. We also sketch remaining challenges and directions for future development.

Probing cytoplasmic and nuclear microRNAs in single living cells via plasmonic affinity sandwich assay.

MicroRNAs (miRNAs) regulate the expression of mRNAs in cells. The determination of subcellular miRNAs in single living cells is essential for understanding the subcellular localizations and functions of miRNAs as well as the microheterogeneity of cells. However, current approaches fail either to keep the cells alive or maintain their original cellular composition or are unable to provide subcellular resolution. Herein, we presented a new approach, called plasmonic affinity sandwich assay (PASA), for rapid and ultrasensitive probing of trace cytoplasmic and nuclear miRNAs in single living cells. It combined efficient in vivo subcellular extraction with ultrasensitive plasmon-enhanced Raman scattering (PERS) detection. By virtue of a micromanipulator, target miRNAs in the cytoplasm and nucleus were first specifically extracted from single living cells by gold thinlayer-coated glass microprobes modified with a half complementary sequence to the target miRNA and then, they were labelled with silver nanotags modified with a Raman reporter and the other half complement. Sandwich-like complexes of extraction sequence-target miRNA-labelling sequence were formed on the extraction microprobe, which were subjected to PERS detection. The subcellular resolution of this approach was confirmed with miR-29b (predominantly localized in the nuclei) and miR-29a (mainly located in the cytoplasm), whereas the quantitative capability was verified with three cytoplasmic miRNAs including miR-21, miR-155 and miR-203. This approach obviated tedious steps such as subcellular fractionation and enzymatic amplification, and it required only 10 min. It could be a promising tool to provide insights into subcellular localizations, functions and microheterogeneity of miRNAs.

Fast probing of glucose and fructose in plant tissues via plasmonic affinity sandwich assay with molecularly-imprinted extraction microprobes.

Determination of specific target compounds in agriculture food and natural plant products is essential for many purposes; however, it is often challenging due to the complexity of the sample matrices. Herein we present a new approach called plasmonic affinity sandwich assay for the facile and rapid probing of glucose and fructose in plant tissues. The approach mainly relies on molecularly imprinted plasmonic extraction microprobes, which were prepared on gold-coated acupuncture needles via boronate affinity controllable oriented surface imprinting with the target monosaccharide as the template molecules. An extraction microprobe was inserted into plant tissues under investigation, which allowed for the specific extraction of glucose or fructose from the tissues. The glucose or fructose molecules extracted on the microprobe were labeled with boronic acid-functionalized Raman-active silver nanoparticles, and thus affinity sandwich complexes were formed on the microprobes. After excess Raman nanotags were washed away, the microprobe was subjected to Raman detection. Upon being irradiated with a laser beam, surface plasmon on the gold-coated microprobes was generated, which further produced plasmon-enhanced Raman scattering of the silver-based nanotags and thereby provided sensitive detection. Apple fruits, which contain abundant glucose and fructose, were used as a model of plant tissues. The approach exhibited high specificity, good sensitivity (limit of detection, 1 µg mL-1), and fast speed (the whole procedure required only 20 min). The spatial distribution profiles of glucose and fructose within an apple were investigated by the developed approach.