Because of the biological importance of thiols, the development of probes for thiols has been an active research area in recent years. In this review, we summarize the results of recent exciting reports regarding thiol-addition reactions and their applications in thiol recognition. The examples reported can be classified into four reaction types including 1,1, 1,2, 1,3, 1,4 addition reactions, according to their addition mechanisms, based on different Michael acceptors. In all cases, the reactions are coupled to color and/or emission changes, although some examples dealing with electrochemical recognition have also been included. The use of thiol-addition reactions is a very simple and straightforward procedure for the preparation of thiol-sensing probes.

Biothiols such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) play crucial roles in maintaining redox homeostasis in biological systems. This Minireview summarizes the most significant current challenges in the field of thiol-reactive probes for biomedical research and diagnostics, emphasizing the needs and opportunities that have been under-investigated by chemists in the selective probe and sensor field. Progress on multiple binding site probes to distinguish Cys, Hcy, and GSH is highlighted as a creative new direction in the field that can enable simultaneous, accurate ratiometric monitoring. New probe design strategies and researcher priorities can better help address current challenges, including the monitoring of disease states such as autism and chronic diseases involving oxidative stress that are characterized by divergent levels of GSH, Cys, and Hcy.

Fluorescent probes, as noninvasive tools for visualizing the metabolism of biomolecules, hold great potential to explore their physiological and pathological processes. For cysteine (Cys), however, none of the reported fluorescent probes could image the metabolic processes in living cells. To achieve this goal, we developed a coumarin derivative based on rational design of the dual recognition sites for Cys and its metabolite, SO. The probe displayed distinct two channels with turn-on fluorescent emission toward Cys and SO, which were successfully applied for imaging both A549 cells and zebrafish. Further, with reversible fluorescent responses toward Cys, the probe could image the enzymatic conversion of Cys to SO in living A549 cells in a ratiometric manner. The present work reports the first probe to image the endogenous generated SO without incubation of the SO donors.

Probes with multiple interaction sites or with single sites promoting tandem reactions target challenging analytes and enable the visualization of in vivo interactions.

Intracellular pH values are some of the most important factors that govern biological processes and the acid-base homeostasis in cells, body fluids and organs sustains the normal operations of the body. Subcellular organelles including the acidic lysosomes and the alkalescent mitochondria undergo various processes such as intracellular digestion, ATP production and apoptosis. Due to their precise imaging capabilities, fluorescent probes have attracted great attention for the illustration of pH modulated processes. Furthermore, based on the unique acidic extracellular environment of acidic lysosomes, fluorescent probes can specifically be activated in cancer cells or tumors. In this review, recently reported lysosome and mitochondria specific pH imaging probes as well as pH-activatable cancer cell-targetable probes have been discussed.

The fabrication of microfluidic chips can be simplified and accelerated by three-dimensional (3D) printing. However, all of the current designs of 3D printed microchips require off-chip bulky equipment to operate, which hindered their applications in the point-of-care (POC) setting. In this work, we demonstrate a new class of movable 3D printed microfluidic chip components, including torque-actuated pump and valve, rotary valve, and pushing valve, which can be operated manually without any off-chip bulky equipment such as syringe pump and gas pressure source. By integrating these components, we developed a user-friendly 3D printed chip that can perform general colorimetric assays. Protein quantification was performed on artificial urine samples as a proof-of-concept model with a smartphone used as the imaging platform. The protein was quantified linearly and was within the physiologically relevant range for humans. We believe that the demonstrated components and designs can expand the functionalities and potential applications of 3D printed microfluidic chip and thus provoke more investigation on manufacturing lab-on-a-chip devices by 3D printers.

Glutathione (GSH) mediates a wide variety of biological events and human diseases. Although it has been the subject of intense study in recent years, a further understanding of its molecular mechanisms and metabolism routes in living cells has remained limited due to a lack of appropriate analytical tools. Sulfur dioxide (SO2), an important metabolite of GSH, is usually associated with the symptoms of neurological disorders, cardiovascular diseases, and lung cancer. Herein, a novel multisignal fluorescent probe was rationally designed and exploited for the simultaneous detection of GSH and its metabolite SO2 via an ICT-FRET synergetic mechanism. The probe shows completely reversed fluorescence responses toward GSH (enhanced red emission) and SO2 (annihilated red fluorescence) with high selectivity and sensitivity. In particular, the probe displayed completely different fluorescent signals (blue-shift) with SO2 in the presence of GSH, thereby allowing the imaging of the metabolism process of GSH to SO2 in two independent channels without spectral cross interference. Given these advantages, this probe has been successfully applied to the real-time monitoring of the SO2 metabolic process in living cells and mice models, and it has thus been found that GSH can metabolize SO2 by enzymatic reaction with TST (thiosulfate sulphurtransferase); additionally, SO2 was transformed into sulfate under SUOX (sulfite oxidase). We anticipate that this research will provide a convenient and efficient tool for understanding the interrelated physiological functions of GSH and SO2 in more biosystems.

Heat stroke (HS) can cause serious organism damage or even death. Early understanding of the mechanism of heat cytotoxicity can prevent or treat heat stroke related diseases. In this work, probe Ly-NT-SP was synthesized, characterized, and used for sulfur dioxide (SO2) detection in lysosomes. PBS solutions of probe Ly-NT-SP at pH 5.0 present a marked broad emission band in the green zone (535 nm). After UV irradiation, the spiropyran group in Ly-NT-SP isomerizes to the merocyanine form (Ly-NT-MR), which presented a weak red-shifted emission at 630 nm. In addition, photocontrolled isomerization of Ly-NT-SP to Ly-NT-MR generated a CC–CN+ fragment able to react, through a Michael addition, with SO2 to yield a highly emissive adduct with a marked fluorescence in the green channel (535 nm). In vitro studies showed a remarkable selectivity of photoactivated Ly-NT-MR to SO2 with a limit of detection as low as 4.7 μM. MTT viability assays demonstrated that the Ly-NT-SP is nontoxic to HeLa cells and can be used to detect SO2 in lysosomes. Taking advantage of this, the sensor is successfully applied to image increasing SO2 values in lysosomes during heat shock for the first time. Moreover, we also confirmed that the increased SO2 can protect the small intestine against damage induced by heat shock through regulating oxidative stress in cells and mice.