Cell-Penetrating Peptide Spirolactam Derivative as a Reversible Fluorescent pH Probe for Live Cell Imaging

A colorless and nonfluorescent spirolactam derivative, RhB-R₁₂K, was synthesized by amide condensation between the carboxyl group of rhodamine B (RhB) and the amino group of cell-penetrating peptide (CPP). The fluorescence intensity of RhB-R₁₂K sharply increased as the pH value decreased from 8.0 to 4.9, demonstrating sensitive and reversible response to intracellular pH distribution. This CPP probe was completely water soluble, had low cytotoxicity, was membrane permeable, and was suitable for pH measurement in various organelles by choosing organelle-specific CPP sequences. Interestingly, CPPs acted not only as carriers but also as indispensable parts of fluorophores here. The presence of active groups on the peptides potentially allows for modification with additional dyes to construct multifunctional and ratiometric probes for cell imaging

Detecting Low-Abundance Molecules at Single-Cell Level by Repeated Ion Accumulation in Ion Trap Mass Spectrometer

Low-abundance metabolites or proteins in single-cell samples are usually undetectable by mass spectrometry (MS) due to the limited amount of substances in single cells. This limitation inspired us to further enhance the sensitivity of commercial mass spectrometers. Herein, we developed a technique named repeated ion accumulation by ion trap MS, which is capable of enhancing the sensitivity by selectively and repeatedly accumulating ions in a linear ion trap for up to 25 cycles. The increase in MS sensitivity was positively correlated with the number of repeated cycles. When ions were repeatedly accumulated for 25 cycles, the sensitivity of adenosine triphosphate detection was increased by 22-fold within 1.8 s. Our technique could stably detect low-abundance ions, especially MS<i>ⁿ</i> ions, at the single-cell level, such as 5-methylcytosine hydrolyzed from sample equivalent to ∼0.2 MCF7 cell. The strategy presented in this study offers the possibility to aid single-cell analysis by enhancing MS detection sensitivity

Multicolor Imaging of Cancer Cells with Fluorophore-Tagged Aptamers for Single Cell Typing

The discrimination of the type of cancer cells remains challenging due to the subtle differences in their expression of membrane receptors. In this work, we developed a multicolor cell imaging method for distinguishing the type of cancer cells with fluorophore-tagged aptamers. We found that the interaction between aptamers and cancer cells was affected by both of the sequence of aptamers and the labeled dyes. As the co-ownership of biomarkers for different cancer cell lines, the fluorophore-tagged aptamers interacted with different cancer cell lines in different degree, resulting in a distinct color to discriminate the type of cancer cells at single cell level. Taking advantage of the cross-reactive ability of the fluorophore-tagged aptamers, we could not only distinguish the cancerous cells quickly from large quantities of noncancerous cells, but also identify the type of the cancerous cells. This work has potential application for cancer diagnostic and therapy in the future

Detecting Low-Abundance Molecules at Single-Cell Level by Repeated Ion Accumulation in Ion Trap Mass Spectrometer

Low-abundance metabolites or proteins in single-cell samples are usually undetectable by mass spectrometry (MS) due to the limited amount of substances in single cells. This limitation inspired us to further enhance the sensitivity of commercial mass spectrometers. Herein, we developed a technique named repeated ion accumulation by ion trap MS, which is capable of enhancing the sensitivity by selectively and repeatedly accumulating ions in a linear ion trap for up to 25 cycles. The increase in MS sensitivity was positively correlated with the number of repeated cycles. When ions were repeatedly accumulated for 25 cycles, the sensitivity of adenosine triphosphate detection was increased by 22-fold within 1.8 s. Our technique could stably detect low-abundance ions, especially MS<i>ⁿ</i> ions, at the single-cell level, such as 5-methylcytosine hydrolyzed from sample equivalent to ∼0.2 MCF7 cell. The strategy presented in this study offers the possibility to aid single-cell analysis by enhancing MS detection sensitivity

Aptamer-Based Plasmonic Sensor Array for Discrimination of Proteins and Cells with the Naked Eye

We developed a colorimetric sensor array with reported protein aptamers as nonspecific receptors. We found that different target proteins could make the aptamer-protected gold nanoparticles (AuNPs) exhibit different aggregation behaviors in the presence of a high concentration salt and cause various color change. On the basis of this phenomenon, we applied a series of reported protein aptamers as a receptor array obtaining a distinct response pattern to each target protein. Seven proteins have been well distinguished with the naked eye at the 50 nM level. Cancerous human cells have also been discriminated from noncancerous cells. This method is simple, label-free, and sensitive. It will broaden the application filed of plasmonic nanoparticle-based sensors and give a new direction of developing sensitive array sensing systems

Protein Discrimination Using Fluorescent Gold Nanoparticles on Plasmonic Substrates

Fluorescent gold nanoparticle (GNP) is an easily synthesized and biocompatible optical platform for sensing and imaging with tunable near-infrared (NIR) emission. However, the relatively low fluorescence (FL) quantum yield limits the further improvement of sensitivity and application. Here, we find that, on plasmonic substrates, the FL intensity of protein-directed synthesized GNPs can be enhanced significantly (∼20-fold). Moreover, protein analytes can interact with GNPs and influence the enhanced fluorescence process so that we can obtain distinct FL image patterns. Then, using the array-based sensing strategy, protein discrimination can be achieved. In our present experiment, five GNPs were used as sensing elements and 10 kinds of proteins at three concentrations (0.2, 0.5, and 1 μM) were successfully identified. This array-based sensing strategy using enhanced-fluorescence from GNPs is highly sensitive and differentiable, expanding the application field of GNPs

Rapid Analysis of Unsaturated Fatty Acids on Paper-Based Analytical Devices via Online Epoxidation and Ambient Mass Spectrometry

In this work, we demonstrate a novel design that allows rapid online identification and quantitation of unsaturated fatty acid CC location isomers via epoxidation and ambient mass spectrometry (MS). Unsaturated fatty acid solution was loaded on a paper strip placed between a low-temperature plasma probe and the inlet of a mass spectrometer. Reactive oxygen species in the plasma promoted epoxidation at the CC, and the product was simultaneously ionized. Upon collision-induced dissociation (CID), the epoxidation product was fragmented to release diagnostic ions specific to the CC location. The whole analytical workflow can be completed within 5 s and is particularly promising for point-of-care (POC) clinical diagnosis, considering its fast, high-throughput nature, and coupling with paper-based analytical devices

In Vivo Nanoelectrospray for the Localization of Bioactive Molecules in Plants by Mass Spectrometry

The method for the localization of bioactive molecules in plants is highly needed since it provides a fundamental prerequisite for understanding their physiological and ecological functions. Here, we propose a simple method termed in vivo nanoelectrospray for the localization of bioactive molecules in plants without sample preparation. A capillary is partly inserted into the plant to sample liquid from a highly located region, and then, a high voltage is applied to the plant to generate an electrospray from the capillary tip for mass spectrometry analysis. Using this method, bioactive molecules such as saccharides, glycoalkaloids, flavonoids, organic acids, and glucosinolates (GLs) are detected in the target regions of living plants or fresh fruits. Original information for endogenous chemicals including liable molecules in plant can be obtained. A sketchy three-dimensional distribution of glycoalkaloids in a cherry tomato has been obtained. The present work provides a powerful tool for the study of bioactive molecules in a living plant by mass spectrometry

Controlling Charge States of Peptides through Inductive Electrospray Ionization Mass Spectrometry

A novel ionization device for controlling the charge states of peptides based on an inductive elecrospray ionization technique was developed. This ion source keeps the major capabilities of electrospray ionization (ESI) which is compatible with liquid separation techniques (such as liquid chromatography (LC) and capillary electrophoresis (CE)) and can be potentially used to control the charge states of peptides accurately by simply varying the AC voltage applied. In comparison with conventional ESI, inductive ESI successfully simplifies the mass spectrum by reducing the charge states of peptide to a singly charged one, as well as eliminating the adduct ions

Hydrogen Sulfide Detection Based on Reflection: From a Poison Test Approach of Ancient China to Single-Cell Accurate Localization

With the inspiration of an ancient Chinese poison test approach, we report a rapid hydrogen sulfide detection strategy in specific areas of live cells using silver needles with good spatial resolution of 2 × 2 μm². Besides the accurate-localization ability, this reflection-based strategy also has attractive merits of convenience and robust response when free pretreatment and short detection time are concerned. The success of endogenous H₂S level evaluation in cellular cytoplasm and nuclear of human A549 cells promises the application potential of our strategy in scientific research and medical diagnosis