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The challenge of "false positive" signals significantly complicates tumor localization and surgical resection, which are pivotal for successful tumor surgeries. Therefore, the development of a method for preoperative tumor localization and intraoperative margin determination holds considerable promise for improving surgical outcomes. In this study, a zero-crosstalk ratiometric tumor-targeting near-infrared (NIR) fluorescent probe was developed for precise cancer diagnosis and intraoperative navigation via NIR fluorescence imaging. This probe integrates a tumor-targeting moiety that selectively homes in on hepatocellular carcinoma cells and exhibits a highly sensitive ratiometric response to γ-glutamyltranspeptidase (GGT), characterized by a substantial emission shift from 830 to 650 nm. This unique NIR ratiometric emission property significantly enhances its effectiveness in early diagnosis and imaging-guided surgery resection. Furthermore, this zero-crosstalk probe successfully monitors GGT activity in blood, cells, and in vivo, endowing its potential for early cancer diagnosis in the clinic. Due to its efficient targeting and sensitive in situ response to GGT in both subcutaneous tumors and orthotopic hepatic tumors, the probe exhibits accurate detection capability for hepatic tumors. Additionally, by leveraging zero-crosstalk ratiometric NIR fluorescence imaging, this tumor-targeting probe can serve as a sprayable tool for delineating tumor margins with precision, thereby furnishing real-time intraoperative imaging guidance during tumor resection surgery.

Ulcerative colitis (UC) is an inflammatory disease that leads to the overexpression of peroxynitrite (ONOO - ). Up to now, it has been a challenge to design a colon-targeted fluorescent probe with a large emission shift that can detect and image ONOO - in UC mice. Here, a fluorophore (pyran-coumarin) is linked with cholic acid to develop a colon-targeted fluorescent probe ( CPC ) for the detection of ONOO - . The fluorescent probe showed strong near-infrared emission at 725 nm. After reacting with ONOO - , it can be observed that the fluorescence intensity at 725 nm decreases obviously, and the signal at 490 nm increases clearly with an obvious emission shift (235 nm). This response mechanism causes the probe to have good sensitivity and selectivity. Additionally, the probe CPC shows good targeting ability for the cells with overexpressingTGR5 receptors and displays good results for detecting exogenous and endogenous ONOO - in the cells. More importantly, the probe CPC can be especially assembled in the colon site to distinguish normal and UC mice and provide significant information for the diagnosis of UC.

While DNA amplifier-built nanobiosensors featuring a DNA polymerase-free catalytic hairpin assembly (CHA) reaction have shown promise in fluorescence imaging assays within live biosystems, challenges persist due to unsatisfactory precision stemming from premature activation, insufficient sensitivity arising from low reaction kinetics, and poor biostability caused by endonuclease degradation. In this research, we aim to tackle these issues. One aspect involves inserting an analyte-binding unit with a photoinduced cleavage bond to enable a light-powered notion. By utilizing 808 nm near-infrared (NIR) light-excited upconversion luminescence as the ultraviolet source, we achieve entirely a controllable sensing event during the biodelivery phase. Another aspect refers to confining the CHA reaction within the finite space of a DNA self-assembled nanocage. Besides the accelerated kinetics (up to 10-fold enhancement) resulting from the nucleic acid restriction behavior, the DNA nanocage further provides a 3D rigid skeleton to reinforce enzymatic resistance. After selecting a short noncoding microRNA (miRNA-21) as the modeled low-abundance sensing analyte, we have verified that the innovative NIR light-powered and DNA nanocage-confined CHA nanobiosensor possesses remarkably high sensitivity and specificity. More importantly, our sensing system demonstrates a robust imaging capability for this cancer-related universal biomarker in live cells and tumor-bearing mouse bodies, showcasing its potential applications in disease analysis.

While three-dimensional (3D) DNA walking amplifiers hold considerable promise in the construction of advanced DNA-based fluorescent biosensors for bioimaging, they encounter certain difficulties such as inadequate sensitivity, premature activation, the need for exogenous propelling forces, and low reaction rates. In this contribution, a variety of profitable solutions have been explored. First, a catalytic hairpin assembly (CHA)-achieved nonenzymatic isothermal nucleic acid amplification is integrated to enhance sensitivity. Subsequently, one DNA component is simply functionalized with a photocleavage-bond to conduct a photoresponsive manner, whereby the target recognition occurs only when the biosensor is exposed to an external ultraviolet light source, overcoming premature activation during biodelivery. Furthermore, a special self-propelling walking mechanism is implemented by reducing biothiols to MnO 2 nanosheets, thereby propelling forces that are self-supplied to a Mn 2+ -reliant DNAzyme. By carrying the biosensing system with a DNA molecular framework to induce a unique concentration localization effect, the nucleic acid contact reaction rate is notably elevated by 6 times. Following these, an ultrasensitive in vitro detection performance with a limit of detection down to 2.89 fM is verified for a cancer-correlated microRNA biomarker (miRNA-21). Of particular importance, our multiple concepts combined 3D DNA walking amplifier that enables highly efficient fluorescence bioimaging in live cells and even bodies, exhibiting a favorable application prospect in disease analysis.

Cysteine is an important biological thiol and is closely related to cancer. It remains a challenge to develop a probe that can provide long-term fluorescence detection and imaging of Cys in cells as well as in living organisms. Here, a solid-state fluorophore HTPQ is combined with an acrylate group to construct a solid-state fluorescent probe HTPQC for Cys recognition. The fluorescence of the probe is quenched when the photoinduced electron transfer (PET) process is turned on and the excited-state intramolecular proton transfer (ESIPT) process is turned off. In the presence of Cys, an obvious solid-state fluorescence signal can be observed. The double quenching mechanism makes the probe HTPQC have the advantages of high sensitivity, good selectivity, and high contrast of biological imaging. Due to low cytotoxicity, the probe HTPQC can be used to detect exogenous and endogenous Cys in living cells and is capable of imaging over long periods of time. By making full use of long wavelengths, the probe can be applied for the detection of Cys levels in tumor mice and equipped with the ability to conduct long-term imaging in vivo.

Cysteine (Cys) is a crucial biological thiol that has a vital function in preserving redox homeostasis in organisms. Studies have shown that Cys is closely related to the development of cancer. Thus, it is necessary to design an efficient method to detect Cys for an effective cancer diagnosis. In this work, a novel tumor-targeting probe (Bio-Cy-S) for dual-modal (NIR fluorescence and photoacoustic) Cys detection is designed. The probe exhibits high selectivity and sensitivity toward Cys. After reaction with Cys, both NIR fluorescence and photoacoustic signals are activated. Bio-Cy-S has been applied for the dual-modal detection of Cys levels in living cells, and it can be used to distinguish normal cells from cancer cells by different Cys levels. In addition, the probe is capable of facilitating dual-modal imaging for monitoring changes in Cys levels in tumor-bearing mice. More importantly, the excellent tumor-targeting ability of the probe greatly improves the signal-to-noise ratio of imaging. To the best of our knowledge, this is the first Cys probe to combine targeting and dual-modal imaging performance for cancer diagnosis.

Adenosine triphosphate (ATP), as an indispensable biomolecule, is the main energy source of cells and is used as a marker for diseases such as cancer and fatty liver. It is of great significance to design a near-infrared fluorescent nanoprobe with excellent performance and apply it to various disease models. Here, a near-infrared fluorescent nanoprobe (ZIF-90@SiR) based on a zeolitic imidazole framework is proposed. The fluorescent nanoprobes are synthesized by encapsulating the dye (SiR) into the framework of ZIF-90. Upon the addition of ATP, the structure of the ZIF-90@SiR nanoprobe is disrupted and SiR is released to generate near-infrared fluorescence at 670 nm. In the process of ATP detection, ZIF-90@SiR shows high sensitivity and good selectivity. Moreover, the ZIF-90@SiR nanoprobe has good biocompatibility due to its low toxicity to cells. It is used for fluorescence imaging of ATP in living cells and thus distinguishing normal cells and cancer cells, as well as distinguishing fatty liver cells. Due to excellent near-infrared fluorescence properties, the ZIF-90@SiR nanoprobe can not only distinguish normal mice and tumor mice but also differentiate normal mice and fatty liver mice for the first time.

Cancer is one of the biggest public enemies of global health with its high morbidity and mortality. Achieving early diagnosis is the most effective means of reducing cancer harm, which requires the use of powerful tools to accurately identify biomarkers. However, most of the reported fluorescent probes for cancer diagnosis can only detect one substance, which makes it difficult to meet the requirements of high accuracy. Here, a fluorescent nanoprobe (CPQ@ZIF-90) for sequential detection of ATP and ONOO - is constructed by encapsulating the ONOO - sensitive unit CPQ within ZIF-90. CPQ@ZIF-90 first reacts with ATP to release CPQ, which greatly enhances the fluorescence at 740 nm. Then, the released CPQ continues to react with ONOO - and is oxidatively cleaved by ONOO - to form a coumarin product with a small π-conjugated structure, which significantly enhances the fluorescence at 510 nm. CPQ@ZIF-90 shows high sensitivity and selectivity for the detection of ATP and then ONOO - . Moreover, CPQ@ZIF-90 has good biocompatibility and successfully realizes the sequential detection of a dual-channel fluorescence change of ATP and ONOO - in living cells and zebrafish and accurately distinguishes normal cells from cancer cells. CPQ@ZIF-90 is expected to be a potential tool for accurate cancer diagnosis through sequential detection of two cancer markers.

A crack-free micrometer-sized compact structure of 1,3,5-tris(4-aminophenyl)benzene-terephthaldehyde-covalent organic frameworks (TAPB-PDA-COFs) was constructed in situ at the tip of a theta micropipette (TMP). The COF-covered theta micropipette (CTP) then created a stable liquid-gas interface inside COF nanochannels, which was utilized to electrochemically analyze the content and distribution of ammonia gas in the microenvironments. The TMP-based electrochemical ammonia sensor (TEAS) shows a high sensing response, with current increasing linearly from 0 to 50,000 ppm ammonia, owing to the absorption of ammonia gas in the solvent meniscus that connects both barrels of the TEAS. The TEAS also exhibits a short response and recovery time of 5 ± 2 s and 6 ± 2 s, respectively. This response of the ammonia sensor is remarkably stable and repeatable, with a relative standard deviation of 6% for 500 ppm ammonia gas dispensing with humidity control. Due to its fast, reproducible, and stable response to ammonia gas, the TEAS was also utilized as a scanning electrochemical microscopy (SECM) probe for imaging the distribution of ammonia gas in a microspace. This study unlocks new possibilities for using a TMP in designing microscale probes for gas sensing and imaging.

It is necessary to explore labeling probes with worthy optical properties and a noninvasive fluorescence imaging manner for stable long-term in situ measuring a single suspension cell. In response to these goals, we herein make a breakthrough on two fronts. On one hand, a co-sensitizer-induced efficient 808 nm near-infrared light-excited luminescence-confined upconversion nanoparticle with a low thermal effect is fabricated by employing a layer-by-layer seed growing approach to develop a sandwich structure, under which the luminescence domain is vastly restricted into an extremely thin inner shell (∼ 2.77 nm) to finally bring about a high-efficiency luminescent resonance energy transfer (LRET) sensing behavior. On the other hand, a self-made optical tweezers integrated upconversion luminescence confocal scanning instrument is applied to enhance the imaging accuracy, after which the liquid viscous force is sufficiently overcome by the resulting single beam gradient force and the analyzed suspension cell is always immobilized to the focal plane to ensure a constant luminescence excitation condition. By making use of a metal ion-dependent DNAzyme and a hairpin DNA strand to design a corresponding LRET sensing system, our nanoprobe shows satisfactory assay performance for two model biomolecules (Ca 2+ and TK1 messenger RNA). Following the optical trapping-assisted imaging, this exceptional measurement method is capable of effectively monitoring the intracellular target changes in different physiological states, endowing a powerful toolbox for single cell analysis.

Hydrogen sulfide (H 2 S) is an important endogenous gas signal molecule in living system, which participates in a variety of physiological processes. Very recent evidence has accumulated to show that endogenous H 2 S is closely associated with various cancers and can be regarded as a biomarker of cancer. Herein, we have constructed a new near-infrared fluorescent probe (DCP-H 2 S) based on isophorone-xanthene dye for sensing hydrogen sulfide (H 2 S). The probe shows remarkable NIR turn-on signal at 770 nm with a large Stokes shift of 200 nm, together with high sensitivity (15-fold) and rapid detection ability for H 2 S (4 min). The probe also possesses excellent selectivity for H 2 S over various other analytes including biothiols containing sulfhydryl (-SH). Moreover, DCP-H 2 S has been successfully applied to visualize endogenous and exogenous H 2 S in living cells (293T, Caco-2 and CT-26 cells). In particular, the excellent ability of DCP-H 2 S to distinguish normal mice and tumor mice is shown, and it is expected to be a powerful tool for detection of H 2 S in cancer diagnosis.

In light of the worthy design flexibility and the good signal amplification capacity, the recently developed DNA motor (especially the DNA walker)-based fluorescent biosensors can offer an admirable choice for realizing bioimaging. However, this attractive biosensing strategy not only has the disadvantage of uncontrollable initiation but also usually demands the supplement of exogenous driving forces. To handle the above obstacles, some rewarding solutions are proposed here. First, on the surface of an 808 nm near-infrared light-excited low-heat upconversion nanoparticle, a special ultraviolet upconversion luminescence-initiated three-dimensional (3D) walking behavior is performed by embedding a photocleavage linker into the sensing elements, and such light-controlled target recognition can perfectly overcome the pre-triggering of the biosensor during the biological delivery to significantly boost the sensing precision. After that, a peculiar self-driven walking pattern is constructed by employing MnO 2 nanosheets as an additional nanovector to physically absorb the sensing frame, for which the reduction of the widespread glutathione in the biological medium can bring about sufficient self-supplied Mn 2+ to guarantee the walking efficiency. By selecting an underlying next-generation broad-spectrum cancer biomarker ( survivin messenger RNA) as the model target, we obtain that the newly formed autonomous 3D DNA motor shows a commendable sensitivity (where the limit of detection is down to 0.51 pM) and even an outstanding specificity for distinguishing single-base mismatching. Beyond this sound assay performance, our sensing approach is capable of working as a powerful imaging platform for accurately operating in various living specimens such as cells and bodies, showing a favorable diagnostic ability for cancer care.

The further development of high-performance fluorescent biosensors to image intracellular microRNAs is beneficial to cancer medicine. By virtue of the need for enzymes and hairpin DNA probes, the entropy-driven reaction-assisted signal amplification strategy has shown an enormous potential to accomplish this task. Nevertheless, this good option still meets with poor biostability, low cell uptake efficiency, and unsatisfactory accuracy. On the basis of these challenges, we put forward here a battery of solving pathways. First, the straight DNA probes are anchored onto the vertexes of dual DNA tetrahedrons, and thus the enzyme resistance of the whole sensing system is observably enhanced. A metal-organic framework (ZIF-8 nanoparticle), which can be effectively dissociated into a weakly acidic environment, then is employed as an additional delivery vehicle to encapsulate such a DNA tetrahedron sustained biosensor and finally bring about a more efficient endocytosis. Last, a kind of photocleavage-linker triggered photoresponsive manner is incorporated to achieve an exceptional precise target identification, by which the biosensor can only be initiated under the irradiation of an externally mild 365 nm ultraviolet light source. In accordance with the above efforts, worthy assay performance toward microRNA-196a has given rise to this newly constructed biosensor, whose sensitivity is down to 2.7 pM and also able to distinguish single-base variation. Beyond that, the amplifier can work as a powerful imaging toolbox to accurately determine the targets in living cells, providing a promising intracellular sensing platform.

Despite that the currently discovered CRISPR-Cas12a system is beneficial for improving the detection accuracy and design flexibility of luminescent biosensors, there are still challenges to extend target species and strengthen adaptability in complicated biological media. To conquer these obstacles, we present here some useful strategies. For the former, the limitation to nucleic acids assay is broken through by introducing a simple functional DNA regulation pathway to activate the unique trans-cleavage effect of this CRISPR system, under which the expected biosensors are capable of effectively transducing a protein (employing dual aptamers) and a metal ion (employing DNAzyme). For the latter, a time-gated luminescence resonance energy transfer imaging manner using a long-persistent nanophosphor as the energy donor is performed to completely eliminate the background interference and a nature-inspired biomimetic periodic chip constructed by photonic crystals is further combined to enhance the persistent luminescence. In line with the above efforts, the improved CRISPR-Cas12a luminescent biosensor not only exhibits a sound analysis performance toward the model targets (carcinoembryonic antigen and Na + ) but also owns a strong anti-interference feature to actualize accurate sensing in human plasma samples, offering a new and applicative analytical tool for laboratory medicine.

Cancer ranks as a leading cause of death in every country of the world. However, if they are discovered early, a lot of cancers can be prevented or cured. Discovering and monitoring cancer markers are the main methods for early diagnosis of cancer. To date, many fluorescent probes designed and used for early cancer diagnosis can only react with a single marker, which always causes insufficient accuracy in complex systems. Herein, a novel near-infrared (NIR) fluorescent probe (CyO-DNP) for the sequential detection of H 2 S and H + is synthesized. In this probe, a heptamethine dye is selected as the fluorophore and a 2,4-dinitrophenyl (DNP) ether is chosen as recognition group. In the presence of H 2 S, CyO-DNP is transformed into CyO, which exhibits an intense fluorescence at 663 nm. Then, H + induces the protonation of CyO to obtain CyOH, and the final fluorescence emission at 793 nm significantly enhances. Owing to the low cytotoxicity and the NIR fluorescence emission, CyO-DNP can sequentially monitor endogenous H 2 S and H + in cancer cells and image exogenous and endogenous H 2 S and H + in mice. It is worth mentioning that CyO-DNP can effectively avoid the false positive signal caused by the liver and kidney and discriminate normal mice and tumor mice accurately. For all we know, CyO-DNP is the first fluorescent probe for early accurate diagnosis of cancer by sequentially detecting H 2 S and H + .

Diabetes is one of the metabolic diseases marked by hyperglycemia and is often accompanied by the occurrence of some complications. As a biomarker of oxidative stress, hydrogen peroxide (H 2 O 2 ) has close association with the occurrence and development of diabetes and its complications. Unfortunately, there is no fluorescent probe reported for imaging H 2 O 2 in diabetic mice. Here, a novel near-infrared (NIR) fluorescent probe named QX-B was designed and synthesized to detect H 2 O 2 . For the probe, the quinolinium-xanthene dye is used as the fluorophore and borate ester is chosen as the response group. After the addition of H 2 O 2 , a strong NIR fluorescence signal at 772 nm is observed. The probe not only shows high sensitivity with 10-fold enhancement but also displays excellent selectivity to H 2 O 2 over other possible interfering species. In the meantime, the possible response mechanism of QX-B toward H 2 O 2 was proposed and verified by the high-performance liquid chromatography (HPLC) experiment, mass spectra (MS) experiment, and density functional theory (DFT) calculation. Furthermore, based on the low cell cytotoxicity of QX-B, it has been applied in imaging exogenous and endogenous H 2 O 2 in HeLa cells, HCT116 cells, 4T1 cells, and zebrafish successfully. More importantly, inspired by the performance of NIR fluorescence, QX-B has been used in monitoring H 2 O 2 in diabetic mice for the first time. This provides very important information for the diagnosis and treatment of diabetes and its complications.

Carbon monoxide (CO), as a crucial gasotransmitter, is endogenously produced by the degradation of heme and plays a critical role in regulating various physiological and pathophysiological processes such as oxidative stress. Thus, an effective fluorescent probe for investigating the relationships between CO and oxidative stress in vivo is necessary. In this paper, a ratiometric near-infrared (NIR) fluorescent probe (CP-CO) based on a coumarin-benzopyran fluorophore for imaging CO is developed. CP-CO itself displays strong coumarin emission due to its spironolactone structure. After the probe is reacted with CO and PdCl 2 , a notable enhancement of emission intensity at 690 nm can be found, which results in an obvious red shift of emission (200 nm). Moreover, CP-CO exhibits high sensitivity toward CO and produces a high enhancement ratio (203-fold). In addition, the probe is applied for ratiometric monitoring of exogenous and endogenous CO levels in HepG2 cells. Furthermore, the fluorescence imaging of CP-CO in zebrafish is performed by two-photon excitation along with excellent penetration ability. Most importantly, CP-CO can visualize the upregulation of CO under lipopolysaccharide (LPS)-induced oxidative stress in a zebrafish model, which vividly reveals its excellent ability in the elucidation of CO function in related biological events.

To enhance signal acquisition stability and diminish background interference for conventional flow bead-based fluorescence detection methods, we demonstrate here an exceptional microfluidic chip assisted platform by integrating near-infrared optical tweezers with upconversion luminescence encoding. For the former, a single 980 nm laser is employed to perform optical trapping and concurrently excite upconversion luminescence, avoiding the fluctuation of the signals and the complexity of the apparatus. By virtue of the favorable optical properties of upconversion nanoparticles (UCNPs), the latter is carried out by employing two-color UCNPs (Er-UCNPs and Tm-UCNPs) with negligible spectral overlaps. With the assistance of the double key techniques, we fabricated complex microbeads referred to a UCNPs-miRNAs-microbead sandwich construct by a one-step nucleic acid hybridization process and then obtained uniform terrace peaks for the automatic and simultaneous quantitative determination of miRNA-205 and miRNA-21 sequences with a detection limit of pM level on the basis of a special home-built flow bead platform. Furthermore, the technique was successfully applied for analyzing complex biological samples such as cell lysates and human tissue lysates, holding certain potential for disease diagnosis. In addition, it is expected that the flow platform can be utilized to investigate many other biomolecules of single cells and to allow analysis of particle heterogeneity in biological fluid by means of optical tweezers.

Liver fibrosis is a major stage in the development of liver disease, and it is important to investigate its pathogenesis for early intervention or even reversal. Recent studies have found that intestinal disease can aggravate liver fibrosis through the role of the "gut-liver axis". Hypoxia is considered to be a typical characteristic of many diseases including ulcerative colitis and liver fibrosis. However, there is no fluorescent probe for hypoxia detection to investigate the "gut-liver axis". Herein we design and synthesize a turn-on fluorescent probe termed Cy-AP, which displays high sensitivity and selectivity to hypoxia given by sodium dithionite (Na 2 S 2 O 4 ) in vitro with near-infrared (NIR) emission (725 nm). The possible response mechanism of Cy-AP toward hypoxia is given and proved though HPLC, MS, and theory calculation. Moreover, on the basis of low cell cytotoxicity, the probe is used in visualizing hypoxia in four cell lines (HepG2, HCT116, HeLa, and MCF-7 cells) by fluorescence imaging, flow cytometry, and 3D imaging. Furthermore, due to its NIR emission, Cy-AP can monitor the hypoxia condition in vivo such as in liver fibrosis mice and ulcerative colitis mice models. In particular, the probe can validate the existence and mechanism of the "gut-liver axis" in vivo by monitoring hypoxia. To the best of our knowledge, this is the first work to give a strategy for studying the "gut-liver axis" by a NIR hypoxia-sensitive fluorescent probe, which will provide some powerful support for delaying the progression of liver fibrosis and thus promoting the treatment of liver disease.

We demonstrate an effective approach to realize active and real-time temperature monitoring around the gold nanobipyramids (AuNBPs)-labeled cancer cell under 808 nm laser irradiation by combining optical tweezers and temperature-sensitive upconversion microparticles (UCMPs). On the one hand, the aptamer-modified AuNBPs that absorb laser at 808 nm not only act as an excellent photothermal reagent but also accurately and specifically bind the target cancer cells. On the other hand, the single optically trapped NaYF 4 :Yb 3+ , Er 3+ UCMPs with a 980 nm laser exhibit temperature-dependent luminescence properties, where the intensity ratio of emission 525 and 547 nm varies with the ambient temperature. Therefore, real-time temperature variation monitoring is performed by 3D manipulation of the trapped single UCMP to control its distance from the AuNBPs-labeled cancer cell while being photothermally killed. The results show distance-related thermal propagation because the temperature increase reaches as high as 10 °C at a distance of 5 μm from the cell, whereas the temperature difference drops rapidly to 5 °C when this distance increases to 15 μm. This approach shows that the photothermal conversion from AuNBPs is sufficient to kill the cancer cells, and the temperature increase can be controlled within the micrometer level at a certain period of time. Overall, we present a micrometer-size thermometer platform and provide an innovative strategy to measure temperature at the micrometer level during photothermal killing of cancer cells.

Azoreductase (AzoR) is an essential reductive enzyme which is closely associated with the intestinal disease such as ulcerative colitis (UC). To date, only a few fluorescent probes for detecting AzoR activity in bacteria or cells have been constructed successfully. It is still challenging to design fluorescent probes for in situ monitoring AzoR in vivo. In this paper, a near-infrared (NIR) fluorescent probe (Cy-Azo) based on hemicyanine is designed and synthesized. The emission of the probe is located at 735 nm in the NIR region, which is favorable for its application in vivo. In addition, Cy-Azo shows high sensitivity to AzoR activity with 17-fold fluorescence enhancement and is particularly selective to AzoR over other enzymes, ions, and amino acids. Meanwhile, a possible response mechanism (the azo group in Cy-Azo is reduced by AzoR and cleaved resulting in the production of Cy-NH 2 ) was proposed and verified by HPLC, MS, and theory calculation. In addition, based on low cell cytotoxicity, Cy-Azo is successfully applied in visualizing the activity of AzoR in two cell lines (HCT116 and HepG2 cells) and three types of bacteria ( E. coli , S. aureus , and P. aeruginosa ). In particular, due to its NIR emission, the probe can monitor AzoR activity in acute and chronic UC mice models. To our knowledge this is the first fluorescent probe for detecting AzoR activity in vivo, which can provide much important information for the diagnosis and treatment of UC.

Herein, a conceptual approach for significantly enhancing a bead-supported assay is proposed. For the fluorescence imaging technology, optical tweezers are introduced to overcome the fluid viscosity interference and immobilize a single tested bead at the laser focus to guarantee a fairly precise imaging condition. For the selection of fluorescent materials and the signal acquisition means, a type of innovative luminescence confined upconversion nanoparticle with a unique sandwich structure is specially designed to act as an efficient energy donor to trigger the luminescent resonance energy transfer (LRET) process. By further combining the double breakthrough with a molecular beacon model, the newly developed detection strategy allows for achieving a pretty high LRET ratio (≈ 88%) to FAM molecules and offering sound assay performance toward miRNA analysis with a detection limit as low as the sub-fM level, and is capable of well identifying single-base mismatching. Besides, this approach not only is able to accurately qualify the low-abundance targets from as few as 30 cancer cells but also can be employed as a valid cancer early warning tool for performing liquid biopsy.

The ability to detect cancer early in an accurate and rapid fashion is of critical importance for cancer diagnosis and accurate resection in surgery. γ-Glutamyltranspeptidase (GGT) is overexpressed in several human cancers, while maintaining a low expression in normal microenvironments, and thus is recognized as an important cancer biomarker. To date, rational design of a zero cross-talk ratiometric near-infrared (NIR) GGT fluorescent probe for efficient cancer diagnosis in various biological samples is still a big challenge. In this work, a zero cross-talk ratiometric NIR GGT fluorescent probe named Cy-GSH is developed. Cy-GSH shows high sensitivity to GGT, which is desired for early cancer diagnosis. Upon additional GGT, a large emission shift from 805 to 640 nm is observed, which is suitable for visualizing deeply located cancer in vivo. In addition, successful monitoring of GGT activity in blood, cells, tissues, and in vivo makes Cy-GSH possess great potential for the clinical cancer early diagnosis. Furthermore, accurately visualizing tumors and metastases in mouse models illuminates that the probe may be a convenient tool for fluorescence-guided cancer surgery. To our knowledge, this is the first report to describe the strategy of a zero cross-talk ratiometric NIR GGT fluorescent probe for early cancer diagnosis and fluorescence-guided surgery.

Analysis and characterization of micro/nano-sized pore structure are critical issues in shale geology and engineering. Scanning electron microscopy (SEM) imaging is one of the most widespread methods for the analysis of the micro/nano-sized pores in shale, but precise identification of the ultrafine pore structure in shale is still a big challenge because shale is so complex that some components may have overlap with pores based on the simple discrimination of gray scale under SEM microscopy. Here, Fe 3 O 4 /Au nanocomposite with magnetic properties is synthesized, characterized, and introduced as a novel pore-marker to improve SEM identification and quantitation of micro/nano-sized pores in shale. Due to the superparamagnetic property, the nanomarker is conveniently controlled by an external magnetic field to fill into pores and offers a sharp contrast imaging between matrix of shale (various gray) and pores (bright), which makes the identification of micro/nano-sized pores in shale much more straightforward and reliable. Furthermore, because gold, as a noble metal, is particularly rare in shale, energy-dispersive X-ray spectroscopy mapping of Au is delicately used to precisely calculate area porosity in shale. Combining with the aforementioned merits of the nanomarker, a precise and practical technique is proposed to promote characterization of micro/nano-sized pores in shale.

The emerging field of plasmonics has promoted applications of optical technology, especially in plasmon-enhanced spectroscopy (PES). However, in plasmon-enhanced fluorescence (PEF), "metal loss" could significantly quench the fluorescence during the process, which dramatically limits its applications in analysis and high-resolution imaging. In this report, silver core silica shell-isolated nanoparticles (Ag@SiO 2 NPs or SHINs) with a tunable thickness of shell are used to investigate the interactions between NPs and emitters by constructing coupling and noncoupling modes. The plasmonic coupling mode between Ag@SiO 2 NPs and Ag film reveals an exceeding integrating spectral intensity enhancement of 330 and about 124 times that of the radiative emission rate acceleration for shell-isolated nanoparticle enhanced phosphorescence (SHINEP). The experimental findings are supported by theoretical calculations using the finite-element method (FEM). Hence, the SHINEP may provide a novel approach for understanding the interaction of plasmon and phosphorescence, and it holds great potential in surface detection analysis and singlet-oxygen-based clinical therapy.

Toehold-mediated DNA strand displacement has proven powerful in the construction of various DNA circuits, DNA machines, and biosensors. So far, many new toehold activation mechanisms have been developed to achieve programmed DNA strand displacement behaviors. However, almost all those toeholds are inflexible via either a covalently attached manner or a complementary hybridization strategy, which limit the versatility of DNA devices. To solve this problem, we developed a new toehold, named "cooperative toehold", to activate DNA strand displacement. On the basis of a base stacking mechanism, the cooperative toehold is comprised of two moieties with completely independent DNA sequences between each other. The cooperative toehold enabled one to continuously tune the rate of DNA strand displacement, as well as more sophisticated strand displacement reactions. The cooperative toehold has also been employed as a universal signal translator for biosensors to qualitatively determine RNA and ATP. Moreover, as a novel specific PCR monitoring system, cooperative toehold-mediated DNA strand displacement can detect the pUC18 plasmid in genomic DNA samples with an aM detection limit.

Intracellular viscosity is an essential microenvironmental parameter and H 2 S is a critical gaseous signaling molecule, which are both related to various physiological processes. It is reported that the change of viscosity and an imbalance of H 2 S production in the mitochondria are both associated with overexpression of amyloid betapeptide (Aβ), which is thought to play a central role in the pathogenesis of Alzheimer's disease (AD). However, to our best knowledge, no fluorescent probe is found for dual detection of mitochondrial viscosity and H 2 S. Herein, a dual-response fluorescent probe (Mito-VS) is designed and synthesized to monitor the level of viscosity and H 2 S, respectively. Mito-VS itself is nonfluorescent due to a free intramolecular rotation between dimethylaniline and pyridine. After the increase of viscosity, the rotation is prohibited and an intense red fluorescence is released. Upon the addition of H 2 S, the probe can react with H 2 S to form compound 3 and a strong green fluorescence can be observed. Moreover, the probe possesses a good mitochondrion-targeting ability and is applied for imaging the change of viscosity on the red channel and visualizing the variation of exogenous and endogenous H 2 S concentration on the green channel in mitochondria. Most importantly, the probe is capable of studying the cross-talk influence of viscosity and H 2 S in mitochondria, which is very beneficial for knowing the pathogenesis of AD.

Establishment of a stable analytical methodology with high-quality results is an urgent need for screening cancer biomarkers in early diagnosis of cancer. In this study, we incorporate holographic optical tweezers with upconversion luminescence encoding to design an imageable suspension array and apply it to conduct the detection of two liver cancer related biomarkers, carcinoembryonic antigen and alpha fetal protein. This bead-based assay is actualized by forming a bead array with holographic optical tweezers and synchronously exciting the upconversion luminescence of corresponding trapped complex beads fabricated with a simple one-step sandwich immunological recognition. Owing to the fact that these flowing beads are stably trapped in the focal plane of the objective lens which tightly converges the array of the laser beams by splitting a 980 nm beam using a diffraction optical element, a fairly stable excitation condition is achieved to provide reliable assay results. By further taking advantage of the eminent encoding capability of upconversion nanoparticles and the extremely low background signals of anti-Stokes luminescence, the two targets are well-identified and simultaneously detected with quite sound sensitivity and specificity. Moreover, the potential on-demand clinical application is presented by employing this approach to respond the targets toward complex matrices such as serum and tissue samples, offering a new alternative for cancer diagnosis technology.

Raman scattering and fluorescence spectroscopy permeate analytic science and are featured in the plasmon-enhanced spectroscopy (PES) family. However, the modest enhancement of plasmon-enhanced fluorescence (PEF) significantly limits the sensitivity in surface analysis and material characterization. Herein, we report a Ag nanoantenna platform, which simultaneously fulfills very strong emission (an optimum average enhancement of 10 5 -fold) and an ultrafast emission rate (∼280-fold) in PES. For applications in surface science, this platform has been examined with a diverse array of fluorophores. Meanwhile, we utilized a finite-element method (FEM) and time-dependent density functional theory (TD-DFT) to comprehensively investigate the mechanism of largely enhanced radiative decay. PES with a shell-isolated Ag nanoantenna will open a wealth of advanced scenarios for ultrasensitive surface analysis.

Analysis of complex pore structure of geomaterials is a fundamental issue in geoscience. Here bifunctional nanoparticles with magnetic and fluorescent properties are introduced as novel markers for optical imaging of pore structure in geomaterials. Using the paramagnetic property, powder of the nanoparticle is driven into pores under an external magnetic field, avoiding a tedious sample preparation and eliminating artificial damage of sample preparation in conventional methods. Meanwhile, the fluorescent nanoparticle marker offers a sharp contrast imaging between the rock matrix (black) and pores (bright) under microscopy. Furthermore, fluorescent nanoparticles with different sizes and colors are designed to demonstrate the potential of the method for describing pore throat sizes. Combining the merits of the paramagnetic and fluorescent properties of nanoparticles, a convenient and practical sample preparation is proposed to promote optical imaging analysis of the pore structure in geomaterials.

Alkaline phosphatase (ALP) is an essential enzyme and widely distributes in a variety of tissues. To date, various nanomaterial and small-molecule fluorescent probes for ALP have been constructed successfully, but the emission wavelengths of these probes are in the ultraviolet or visible range, which is not beneficial for bioimaging. Herein, a hemicyanine-based near-infrared (NIR) fluorescent probe named CyP is first synthesized and used to detect ALP activity. The characteristics of probe CyP are as follows: (1) The probe possesses a facile structure, which can be obtained by easy synthetic steps. (2) The fluorescence emission of the sensing system is at 738 nm belonging to NIR region, which is suitable for bioimaging in vivo. (3) The probe exhibits high sensitivity to ALP with 10-fold fluorescence enhancement and low detection limit (0.003 U/mL) can match the level of ALP in vivo. (4) The fluorescent change of the probe is attributed to the fact that ALP-catalyzed cleavage of the phosphate group in CyP induces the transformation of CyP (fluorescence off) into CyOH (fluorescence on), which is proved by HPLC, 31 P NMR, MS, and DFT calculation. (5) The NIR fluorescent probe is applied for the detection of endogenous ALP activity in various biological samples such as cell, tissue, and living animal with satisfactory results.

Adenosine triphosphate (ATP) is mainly produced in the mitochondrion and used as a universal energy source for various cellular events. Various fluorescent probes for ATP have been established successfully, but most of them are not appropriate for monitoring the fluctuation of the mitochondrial ATP level. Herein, a fluorescent probe named Mito-Rh is first synthesized and used to recognize ATP in mitochondrion. In the probe, rhodamine, diethylenetriamine, and triphenylphosphonium are selected as fluorophore, reaction site, and mitochondrion-targeting group, respectively. Probe Mito-Rh shows high sensitivity to ATP with 81-fold fluorescence enhancement, and the detection range (0.1-10 mM) can match the concentration level of ATP in the mitochondrion. Moreover, Mito-Rh provides excellent selectivity toward ATP over other biological anions (ADP, AMP, GTP, CTP, UTP) owing to a concurrent effect of dual recognition sites (hydrogen bond and π-π stacking). In particular, the probe can localize in mitochondrion specifically and demonstrates utility in the real-time detection of mitochondrial ATP concentration changes.

Glutathione (GSH), cysteine (Cys), and homocysteine (Hcy) are small-molecular biothiols that play key roles in various biological systems. Among these biothiols, GSH is the most abundant intracellular thiol. Until now, a small number of the near-infrared (NIR) fluorescent probes have been designed for the detection of GSH. Unfortunately, most of these NIR probes are based on cyanine dyes, which generally suffer low fluorescence quantum yield (Φ < 0.25), which are not suitable for bioimaging. In addition, some probes are difficult to effectively distinguish GSH from Cys and Hcy. In this work, an NIR fluorescent probe with high fluorescence quantum yield is developed by introducing a rigid coplanar structure such as rhodamine dyes, and the NIR probe (CyR) with spirolactam structure is first synthesized and used to recognize GSH. The characteristics of this NIR probe are as follows: (1) probe CyR exhibits high fluorescence quantum yield (Φ = 0.43) after the addition of GSH and high sensitivity toward GSH with 75-fold fluorescence enhancement. (2) The probe is highly selective, which will not interfere with the other biological thiols (Cys, Hcy) and amino acids. (3) A possible reaction mechanism of the NIR probe CyR and GSH (Cys, Hcy) can be proposed and proved by 1 H NMR, 13 C NMR, and MS (mass spectra). (4) The NIR probe displays selective detection of GSH in biological samples such as living cells and tissues.

We present an analytical platform by combining near-infrared optical tweezers with two-photon excitation for fluorescence detection of H5N1 virus gene sequences. A heterogeneous enrichment strategy, which involved polystyrene (PS) microsphere and quantum dots (QDs), was adopted. The final hybrid-conjugate microspheres were prepared by a facile one-step hybridization procedure by using PS microspheres capturing target DNA and QDs tagging, respectively. Quantitative detection was achieved by the optical tweezers setup with a low-cost 1064 nm nanosecond pulse laser for both optical trapping and two-photon excitation for the same hybrid-conjugate microsphere. The detection limits for both neuraminidase (NA) gene sequences and hemagglutinin (HA) gene sequences are 16-19 pM with good selectivity for one-base mismatch, which is approximately 1 order of magnitude lower than the most existing fluorescence-based analysis method. Besides, because of the fact that only signal from the trapped particle is detected upon two-photon excitation, this approach showed extremely low background in fluorescence detection and was successfully applied to directly detect target DNA in human whole serum without any separation steps and the corresponding results are very close to that in buffer solution, indicating the strong anti-interference ability of this method. Therefore, it can be expected to be an emerging alternative for straightforward detecting target species in complex samples with a simple procedure and high-throughput.

A new ionic current rectification device responsive to a broad range of pH stimuli has been fabricated using porous anodic alumina membrane with tailor-made branching nanochannel array. The asymmetric geometry is achieved by changing oxidation voltage using a simple two-step anodization process. Due to the protonation/deprotonation of the intrinsic hydroxyl groups upon solution pH variation, the nanochannels array-based device is able to regulate ionic current rectification properties. The ion rectification ratio of the device is mainly determined by the branching size and surface charges, which is also confirmed by theoretical simulations. In addition, theoretical simulations show that the slight difference in ion rectification ratio for the nanochannel devices with different branching numbers is due to the propagation of concentration polarization. Three dimensional simulations clearly show the nonuniform concentration profiles in stem nanochannel near the junction interface due to the presence of branching nanochannels. The present ionic device is promising for biosensing, molecular transport and separation, and drug delivery in confined environments.

In this paper, we unveil a novel naphthalimide-porphyrin hybrid based fluorescence probe (1) for ratiometric detection of Hg(2+) in aqueous solution and living cells. The ratiometric signal change of the probe is based on a carefully predesigned molecule containing two independent Hg(2+)-sensitive fluorophores with their maximal excitation wavelengths located at the same range, which shows reversibly specific ratiometric fluorescence responses induced by Hg(2+). In the new developed sensing system, the emissions of the two fluorophores are well-resolved with a 125 nm difference between two emission maxima, which can avoid the emission spectra overlap problem generally met by spectra-shift type probes and is especially favorable for ratiometric imaging intracellular Hg(2+). It also benefits from a large range of emission ratios and thereby a high sensitivity for Hg(2+) detection. Under optimized experimental conditions, the probe exhibits a stable response for Hg(2+) over a concentration range from 1.0 x 10(-7) to 5.0 x 10(-5) M, with a detection limit of 2.0 x 10(-8) M. The response of the probe toward Hg(2+) is reversible and fast (response time less than 2 min). Most importantly, the ratiometric fluorescence changes of the probe are remarkably specific for Hg(2+) in the presence of other abundant cellular metal ions (i.e., Na(+), K(+), Mg(2+), and Ca(2+)), essential transition metal ions in cells (such as Zn(2+), Fe(3+), Fe(2+), Cu(2+), Mn(2+), Co(2+), and Ni(2+)), and environmentally relevant heavy metal ions (Ag(+), Pb(2+), Cr(3+), and Cd(2+)), which meets the selective requirements for biomedical and environmental monitoring application. The recovery test of Hg(2+) in real water samples demonstrates the feasibility of the designed sensing system for Hg(2+) assay in practical samples. It has also been used for ratiometric imaging of Hg(2+) in living cells with satisfying resolution, which indicates that our novel designed probe has effectively avoided the general emission spectra overlap problem of other ratiometric probes.

The design and synthesis of a novel rhodamine spirolactam derivative and its application in fluorescent detections of Cu(2+) in aqueous solution and living cells are reported. The signal change of the chemosensor is based on a specific metal ion induced reversible ring-opening mechanism of the rhodamine spirolactam. It exhibits a highly sensitive "turn-on" fluorescent response toward Cu(2+) in aqueous solution with an 80-fold fluorescence intensity enhancement under 10 equiv of Cu(2+) added. This indicates that the synthesized chemosensor effectively avoided the fluorescence quenching for the paramagnetic nature of Cu(2+) via its strong binding capability toward Cu(2+). With the experimental conditions optimized, the probe exhibits a dynamic response range for Cu(2+) from 8.0 x 10(-7) to 1.0 x 10(-5) M, with a detection limit of 3.0 x 10(-7) M. The response of the chemosensor for Cu(2+) is instantaneous and reversible. Most importantly, both the color and fluorescence changes of the chemosensor are remarkably specific for Cu(2+) in the presence of other heavy and transition metal ions (even those that exist in high concentration), which meet the selective requirements for biomedical and environmental monitoring application. The proposed chemosensor has been used for direct measurement of Cu(2+) content in river water samples and imaging of Cu(2+) in living cells with satisfying results, which further demonstrates its value of practical applications in environmental and biological systems.