Search

Sensitive and specific assay of microRNAs (miRNAs) is beneficial to early disease screening. Herein, we for the first time proposed clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a-mediated photoelectrochemical biosensors for the direct assay of miRNA-21. In this study, compared with traditional nucleic acid-based signal amplification strategies, the CRISPR/Cas13a system can greatly improve the specificity and sensitivity of target determination due to its accurate recognition and high-efficient

Tumor-specific imaging is a major challenge in clinical tumor resection. To overcome this problem, several activatable probes have been developed for use in tumor imaging. However, most of these probes are activated based on a single-factor stimulation and are irreversible. Therefore, false signals that make tumor-specific imaging difficult are easily generated. We have developed a new dual-stimulus responsive near-infrared (NIR) reversible adenosine 5'-triphosphate (ATP)-pH probe for fluorescence and photoacoustic ratiometric imaging of tumors. Since the H+ and ATP content is significantly higher in the tumor microenvironment than that in normal tissues, the Forster resonance energy transfer-based probe ATP-pH was constructed with silicon rhodamine as the donor, CS dye as the acceptor, and ATP/H+ recognition units that could only be activated when both H+ and ATP were connected to the acceptor. The ATP-pH probe is reversibly activated by both the H+ and ATP, which effectively reduces the cumulative response of the probe in circulation after intravenous injection. Further, the NIR ratiometric property of the probe makes it suitable for in vivo imaging. Finally, our probe was successfully utilized in ratiometric photoacoustic and fluorescence tumor imaging and ratiometric fluorescence imaging-guided tumor resection.

Long-term specific tracing of the fibroblast activation protein (FAP) has been of great importance because it is heavily expressed by stromal fibroblasts of multiple diseases, and several disorders associated with FAP are chronical. Bioluminescence (BL) imaging has its advantages to detect FAP in vivo since no external excitation is required, but the current FAP-responsive BL probe was constructed by covalently masking the firefly luciferase substrate and easily secreted out from the animal, resulting in transient BL imaging of FAP. To circumvent this problem, a peptide-linked amphiphilic block copolymer-based probe (PABC) was developed and applied to the long-lasting BL image of FAP in vivo. For this purpose, an amphiphilic block copolymer containing an FAP-responsive peptide was fabricated to self-assemble into micelles, which act as a depot to load amounts of d-luciferin for constructing the BL probe. Upon reaction with FAP, the micelle would be destroyed to release the internal d-luciferin for BL emission by a luciferase-catalyzed reaction. By virtue of the high loading capability of micelles, the FAP was determined from 0.5 to 10 ng/mL with a detection limit of 0.105 ng/mL, and the high sensitivity makes the PABC capable of distinguishing cancer cells from normal ones. Importantly, compared with free d-luciferin, PABC can be used to persistently image the FAP in living cells and in vivo. This characteristic of long-lasting specific tracing of the FAP makes us envision that this BL probe could be used for screening of FAP inhibitors and diagnosing various FAP-related diseases in future.

Tyrosinase (TYR) is a crucial enzyme involved in melanogenesis, and its overexpression is closely associated with melanoma. To precisely monitor intracellular TYR activity, remote control of a molecule imaging tool is highly meaningful but remains to be explored. In this work, we present the first photocaged tyrosinase fluorogenic probe by caging the substrate of the enzymatic probe with a photolabile group. Because of the sequential light and enzyme-activation feature, this probe exhibits photocontrollable "turn on" response toward TYR with good selectivity and high sensitivity (detection limit: 0.08 U/mL). Fluorescence imaging results validate that the caged probe possesses the capability of visualizing intracellular endogenous tyrosinase activity in a photocontrol fashion, thus offering a promising molecule imaging tool for investigating TYR-related physiological function and pathological role. Moreover, our sequential activation strategy has great potential for developing more photocontrollable enzymatic fluorogenic probes with spatiotemporal resolution.

Accurate and sensitive imaging of hypoxia associated with inflammatory bowel disease (IBD) is significant for the precise diagnosis and treatment of this disease, but it remains a challenge for traditional hypoxia-activatable fluorescence probes because of a more moderate hypoxic state during IBD than under other pathological conditions. To address this issue, herein, we designed a hypoxia-activatable and cytoplasmic protein-powered fluorescence cascade amplifier, named HCFA, to image hypoxia associated with IBD in vivo. In our design, a 4-aminobenzoic acid (azo)-modified mesoporous silica nanoparticle (MSN) was used as a container to load black hole quencher 2 (BHQ2) and cytoplasmic protein-binding squarylium dye (SQ); then, the β-cyclodextrin polymer (β-CDP) combined with azo through a host-guest interaction to form HCFA. Upon passive stagnation in the inflamed tissue of IBD, the azo band would be cleaved under a hypoxic microenvironment, and SQ was released to activate the fluorescence of HCFA. Moreover, the unconstrained SQ can bind with cytoplasmic protein to exhibit drastic fluorescence intensity enhancement, realizing the fluorescence signal amplification for imaging of hypoxia. When one takes advantage of the large load capacity of MSN and the unique property of SQ, HCFA can sense oxygen levels in the range of 0% to 10%. Meanwhile, the fluorescence imaging results demonstrate that HCFA can sensitively distinguish different levels of cellular hypoxia and monitor the variations of hypoxia in vivo, highlighting HCFA as a promising tool for the detection of hypoxia associated with IBD.

Tiagabine hydrochloride (TGB) is a clinically frequently used drug for anticonvulsion and reducing epileptic frequency. Over administration of TGB could bring about adverse effects, such as speech disorder, depression, and even suicidal tendencies. Therefore, accessible and sensitive assay for analysis of TGB becomes an urgent need toward guiding clinical medication. Here, we present the first report on fluorescence turn-on detection of TGB in urine testing. In this protocol, a fluorescent dye, perylene tetracarboxylic acid imide derivative (PTAI), is found specifically occupying the Sudlow site II of human serum albumin (HSA) and displays a new phenomenon of binding-induced quenching (BIQ). In presence of TGB, competitive binding of the TGB to the site II of HSA will trigger release of PTAI, thus successfully lighting up the fluorescence of PTAI. This label-free assay enjoys a broader working range (1-350 μM) and lower detection limit (0.218 μM) than the traditional liquid chromatography method and is uninterfered by the miscellaneous in the artificial urine. The BIQ probe highlights the merits of HSA as a quencher and a molecular recognition unit, and it opens up a way for studying drug-HSA interaction mechanism and noninvasive pharmaceutical testing.

On the basis of a target microRNA (miRNA)-responsive DNA hydrogel, a novel surface-enhanced Raman scattering (SERS) sensor array with nine sensor units that can detect multiple cancer-related miRNAs in one sample was developed. The target miRNA-responsive DNA hydrogel was first formed in each sensor unit to realize the construction of the DNA hydrogel-based SERS sensor array. Initially, because of the blocking of the streptavidin (SA)-modified sensor units by the formed DNA hydrogel, the SERS tags (biotin/4-mercaptobenzonitrile-functionalized AuAg alloy nanoparticles (B/M-AuAgNPs)) could not pass through the hydrogel and bind to the SA-modified sensor surface; thus, obvious Raman signals could not be observed. After the introduction of the target miRNA, DNA hydrogels of the corresponding sensor unit were disintegrated accordingly, and SERS tags were able to pass through the hydrogel to be captured onto the SA-modified detection surface, thus resulting in strong Raman signals and the detection of target miRNA. The assay is validated under clean buffer conditions as well as in serum. This target miRNA-responsive DNA hydrogel-based SERS sensor array has attractive application prospects in cancer typing via blood miRNA measurements.

Herein, we describe a novel two-photon excitation/red emission-based ratiometric pH nanosensor consisting of a pH-sensitive two-photon dye and Tm3+-doped upconversion nanoparticles (UCNP). The fluorescence emission ratio between the dye (610 nm) and UCNPs (810 nm) (I610/I810) provides a linear indicator of pH values in the range from pH 4.0 to 6.5 with high sensitivity. These nanoprobes selectively accumulate in the lysosomes of cells, making them suitable for lysosomal pH tracking. This pH nanoprobe has been successfully applied in visualizing chemically stimulated changes of intracellular pH in living cells and tissues.

Fluorescence amplification is critical for in situ and real-time detection of intracellular low abundance biological species. However, current intracellular amplification techniques mainly rely on synthetic nucleic acid-based nanodevices, manipulating them in living cells remains challenging. To solve this problem, herein, a new signal amplification concept named cytoplasmic protein-powered in situ fluorescence amplification (CPFA) is proposed. CPFA takes cytoplasmic protein as cell-self-power for signal amplification enabling it to operate in living cells. To establish a prototype of CPFA, an amplifiable sensor for hydroxyl radicals (•OH) was designed by entrapping the screened cytoplasmic protein-enhanced fluorophore (PBF1) inside mesoporous silica (MSN) nanocontainer with ssDNA/PTAD-based signal switch. When encountered with •OH in living cells, the ssDNA was cleaved to separate PTAD from MSN, liberating multiple copies of the loaded PBF1 to light up the fluorescence. Furthermore, these released PBF1 molecules can instantly bind with cytoplasmic proteins to amplify their fluorescence signals. Take advantage of this two-stage amplification mode, the sensor in response to •OH exhibited remarkable fluorescence enhancement (near 400-fold) in cell lysates, and the •OH was linearly determined from 0 to 800 nM with a detection limit of 6.4 pM. Moreover, this sensor can track basal level and fluctuation of •OH in living cells on account of its high sensitivity. To our knowledge, this is the first effort to use cytoplasmic protein for amplifying detection signals, which will provide a new dimension to current methodologies for low-abundance biomarkers discovery and regulation for chemical biology and medical diagnostics.

Tumor-specific imaging is a major challenge in clinical tumor resection. To overcome this problem, several activatable probes have been developed for use in tumor imaging. However, most of these probes are activated based on a single-factor stimulation and are irreversible. Therefore, false signals that make tumor-specific imaging difficult are easily generated. We have developed a new dual-stimulus responsive near-infrared (NIR) reversible adenosine 5'-triphosphate (ATP)-pH probe for fluorescence and photoacoustic ratiometric imaging of tumors. Since the H

A new triplex-functionalized DNA tetrahedral nanoprobe is proposed herein for monitoring pH and messenger RNA (mRNA) in living cells. Different from traditional DNA tetrahedron-based nanoprobes, DNA triplex was employed to serve as important conformational conversion elements. Inspired by the low extracellular pH in tumor cells, the mRNA-targeted H1 and H2 were stably assembled on the extended short hairpin probes of DNA tetrahedron via Hoogsteen bonding to form DNA triplex. Due to the high intracellular pH and presence of target mRNA, hybridization chain reaction (HCR) was triggered between H1 and H2 which were released from the dissociation of DNA triplex, and the generated long double-stranded DNA activated a Föster resonance energy transfer (FRET) signal indicating target mRNA expression even at very low contents. By combining the distinguishing feature of DNA triplex structure (pH-responsive) and HCR (signal amplification), sensitive imaging of intracellular pH and tumor-related mRNA can be realized. As a further application, dynamic imaging of intracellular pH and mRNA during "mitochondria-dependent" pathway apoptosis was successfully achieved in human breast cancer cells, which indicated huge potential of our proposed nanoprobe in early diagnosis and treatment of diseases.

Fluorescence molecular imaging has attracted increasing attention due to its various advantages. Lots of fluorophores have been developed to meet various molecular imaging needs. However, it is still inconvenient due to the lack of excellent fluorophores with an optically tunable group for biological molecular imaging. Here a new platform of a versatile long wavelength fluorophore with an optically tunable hydroxyl group was successfully developed by regulating molecular planarity and the twisted intramolecular charge transfer effect with a protected and deprotected hydroxyl group approach via "step by step" modifying strategy. As an excellent representative of this new type of fluorophore, LDOH-4 possesses good chemical and optical properties and shows a potential application prospect. As a proof-of-concept, a nitroreductase-activated TP fluorescent probe LDO-NTR was designed, which not only sensitively recognizes NTR with more than 310-fold response signal enhancement in vitro but also tracks NTR in a hypoxia tumor mouse model in vivo by using two-photon imaging. It is hopeful that the long wavelength fluorophore with the optically tunable hydroxyl group can serve as a useful platform to extend capable detection tools in biological chemistry and biomedical applications.

Hypoxia-induced cell apoptosis is closely related to degenerative diseases, autoimmune disorders, and tumor disease. In the process of apoptosis, the release of cytochrome c (Cyt c) is deemed to be a critical factor of the intrinsic pathway. Strategies for tracking Cyt c release in living cells based on the subcellular localization have been proposed recently. However, they are inherently lack of specificity for distinguishing the release of Cyt c in apoptotic process induced by hypoxia from other stimulus. In this paper, an azoreductase and target simultaneously activated fluorescent aptameric nanosensor integrating gold nanoparticles (AuNPs) and Cyt c-targeted aptamer-consisted double-stranded DNA hybridization complex (DSDHC) was proposed. It is worth noting that the employment of azobenzene moiety labeled on the DSDHC first ensured the aptameric nanosensor could be conjugated to the surface of AuNPs and then specifically reduced by hypoxia-related azoreductase. Upon Cyt c released from mitochondrion under hypoxia, the competitive displacement of Cyt c subsequently activated the fluorescence of the aptameric nanosensor and the fluorescence enhancement depended principally on the content of Cyt c release. Inspired by this, a new strategy for quantitative analysis and in situ imaging of Cyt c under hypoxic condition was proposed. The high spatial resolution monitoring of the dynamics of Cyt c release under hypoxia will offer a potentially rich opportunity to understand the apoptotic mechanism under hypoxic conditions, thus further facilitating risk assessment and risk reduction for hypoxic environments.

It is of scientific significance to explore the intricate relationship between two crucial gasotransmitters nitric oxide (NO) and hydrogen sulfide (H2S) because they exert similar and interdependent biological actions within the living organisms. Nevertheless, visualization of the NO/H2S crosstalk using effective molecular imaging tools remains challenging. To address this issue, and given that nitroxyl (HNO) has been implicated as the interdependent production of NO and H2S via a network of cascading chemical reactions, we herein design a ratiometric two-photon fluorescent probe for HNO, termed TP-Rho-HNO, which consists of benzo[h]chromene-rhodol scaffold as two-photon energy transfer cassette with phosphine moiety as specific HNO recognition unit. The newly proposed probe has been successfully applied in ratiometric two-photon bioimaging of endogenous HNO derived from NO and H2S interaction in the human umbilical vein cells (HUVECs) and as well as in rat brain tissues. Intriguingly, the imaging results...

Mitophagy, as an evolutionarily conserved cellular process, plays a crucial role in preserving cellular metabolism and physiology. Various microenvironment alterations assigned to mitophagy including pH, polarity, and deregulated biomarkers are increasingly understood. However, mitophagy-specific viscosity dynamic in live cells remains a mystery and needs to be explored. Here, a water-soluble mitochondria-targetable molecular rotor, ethyl-4-[3,6-bis(1-methyl-4-vinylpyridium iodine)-9 H-carbazol-9-yl)] butanoate (BMVC), was exploited as a fluorescent viscosimeter for imaging viscosity variation during mitophagy. This probe contains two positively charged 1-methyl-4-vinylpyridium components as the rotors, whose rotation will be hindered with the increase of environmental viscosity, resulting in enhancement of fluorescence emission. The results demonstrated that this probe operates well in a mitochondrial microenvironment and displays an off-on fluorescence response to viscosity. By virtue of this probe, new discoveries such as the mitochondrial viscosity will increase during mitophagy are elaborated. The real-time visualization of the mitophagy process under nutrient starvation conditions was also proposed and actualized. We expect this probe would be a robust tool in the pathogenic mechanism research of mitochondrial diseases.

Hyaluronidase has two cruical isoforms, hyaluronidase-1 (Hyal-1) and hyaluronidase-2 (Hyal-2), which are essential for cellular hyaluronic acid (HA) catabolism to generate different-sized oligosaccharide fragments for performing different physiological functions. In particular, Hyal-1 is the major tumor-derived hyaluronidase. Thus, specific detection of one hyaluronidase isoform, especially Hyal-1, in live cells is of scientific significance but remains challenging. Herein, by use of differentiated tolerance capability of an amphiphilic HA-based nanoassembly to Hyal-1 and Hyal-2, we rationally design a Hyal-1 specific nanosensor, consisting of cholesterylamine-modified HA nanoassembly (CHA) and RNA-binding fluorophores (RBF). The RBF molecules were entrapped in CHA to switch off their fluorescence via aggregation caused quenching. However, CHA can be disassembled by Hyal-1 to release RBF, resulting in fluorescence activation. Moreover, the fluorescence of the released RBF is further enhanced by cytoplasm RNA. Owing to this cascade signal amplification, this nanosensor RBF@CHA displays a significant change of signal-to-background-noise ratio (120-fold) toward 16 μg/mL Hyal-1 in cellular lysates. In contrast, it is resistant to Hyal-2. By virtue of its selective and sensitive characteristics under a complicated matrix, RBF@CHA had been successfully applied for specifically visualizing Hyal-1 over Hyal-2 inside live cells for the first time, detecting a low level of intracellular Hyal-1 and distinguishing normal and cancer cells with different expressions of Hyal-1. This approach would be useful to better understand biological functions and related diseases of intracellular Hyal-1.

Febrile seizure (FS), a frequently encountered seizure disorder in pediatric populations, can cause hippocampus damage. It has been elucidated that sulfur dioxide (SO2) content is overproduced during the development of FS and related brain injury. Thus, monitoring in situ the level of endogenous SO2 in FS-related models is helpful to estimate the pathogenesis of FS-induced brain injury, but the effect detection method remains to be explored. Herein, we developed a two-photon energy transfer cassette based on an acedan–anthocyanidin scaffold, TP-Ratio-SO2, allowing us to achieve this purpose. TP-Ratio-SO2 specifically responds to SO2 derivatives (HSO3–/SO32–) in an ultrafast fashion (less than 3 s), and HSO3–/SO32– can be sensitively determined with a detection limit of 26 nM. Moreover, it exhibits significant changes in two well-resolved fluorescence emissions (Δλ = 140 nm) by reacting with HSO3–/SO32–, behaving as a ratiometric fluorescent sensor. Importantly, ratiometric imaging of endogenous SO2 deriva...

G-quadruplex analogues have been widely used as molecular tools for detection of potassium ion (K(+)). However, interference from a higher concentration of sodium ion (Na(+)), enzymatic degradation of the oligonucleotide, and background absorption and fluorescence of blood samples have all limited the use of G-quadruplex for direct detection of K(+) in blood samples. Here, we reported, for the first time, an intermolecular G-quadruplex-based assay capable of direct fluorescent detection of blood K(+). Increased stringency of intermolecular G-quadruplex formation based on our screened G-rich oligonucleotide (5'-TGAGGGA GGGG-3') provided the necessary selectivity for K(+) against Na(+) at physiological ion level. To increase long-term stability of oligonucleotide in blood, the screened oligonucleotide was modified with an inverted thymine nucleotide whose 3'-terminus was connected to the 3'-terminus of the upstream nucleotide, acting as a blocking group to greatly improve antinuclease stability. Lastly, to avoid interference from background absorption and autofluorescence of blood, a G-quadruplex-binding, two-photon-excited ligand, EBMVC-B, was synthesized and chosen as the fluorescence reporter. Thus, based on selective K(+) ion-induced formation of intermolecular G-quadruplex and EBMVC-B binding, this approach could linearly respond to K(+) from 0.5 to 10 mM, which matches quite well with the physiologically relevant concentration of blood K(+). Moreover, the system was highly selective for K(+) against other metal ions, including Na(+), Ca(2+), Mg(2+), Zn(2+) common in blood. The practical application was demonstrated by direct detection of K(+) from real blood samples by two-photon fluorescence technology. To the best of our knowledge, this is the first attempt to exploit molecular G-quadruplex-based fluorescent sensing for direct assay of blood target. As such, we expect that it will promote the design and practical application of similar DNA-based sensors in complex real systems.

By virtue of its high sensitivity and rapidity, Fenton reaction has been demonstrated as a powerful tool for in vitro biochemical analysis; however, in vivo applications of Fenton reaction still remain to be exploited. Herein, we report, for the first time, the design, formation and testing of Fenton reaction for in vivo fluorescence imaging of hydrogen peroxide (H2O2). To realize in vivo fluorescence imaging of H2O2 via Fenton reaction, a functional nanosphere, Fc@MSN-FDNA/PTAD, is fabricated from mesoporous silica nanoparticle (MSN), a Fenton reagent of ferrocene (Fc), ROX-labeled DNA (FDNA), and a cationic perylene derivative (PTAD). The ferrocene molecules are locked in the pore entrances of MSN, and exterior of MSN is covalently immobilized with FDNA. As a key part, PTAD acts as not only the gatekeeper of MSN but also the efficient quencher of ROX. H2O2 can permeate into the nanosphere and react with ferrocene to product hydroxyl radical (·OH) via Fenton reaction, which cleaves FDNA to detach ROX from PTAD, thus in turn, lights the ROX fluorescence. Under physiological condition, H2O2 can be determined from 5.0 nM to 1.0 μM with a detection limit of 2.4 nM. Because of the rapid kinetics of Fenton reaction and high specificity for H2O2, the proposed method meets the requirement for real applications. The feasibility of Fc@MSN-FDNA/PTAD for in vivo applications is demonstrated for fluorescence imaging of exogenous and endogenous H2O2 in cells and mice. We expect that this work will not only contribute to the H2O2-releated studies but also open up a new way to exploit in vivo Fenton reaction for biochemical research.

Methylmercury (CH3Hg+), the common organic source of mercury, is well-known as one of the most toxic compounds that is more toxic than inorganic or elemental mercury. In seabeds, the deposited Hg2+ ions are converted into CH3Hg+ by bacteria, where they are subsequently consumed and bioaccumulated in the tissue of fish, and finally, to enter the human diet, causing severe health problems. Therefore, sensitive and selective detection of bioaccumulation of CH3Hg+ in fish samples is desirable. However, selective assay of CH3Hg+ in the mercury-containing samples has been seriously hampered by the difficulty to distinguish CH3Hg+ from ionic mercury. We report here that metal amalgamation, a natural phenomenon occurring between mercury atoms and certain metal atoms, combining with DNA-protected silver nanoparticles, can be used to detect CH3Hg+ with high sensitivity and superior selectivity over Hg2+ and other heavy metals. In our proposed approach, discrimination between CH3Hg+ and Hg2+ ions was realized by for...

Accurate determination of intracellular glucose is very important for exploring its chemical and biological functions in metabolism events of living cells. In this paper, we developed a new noninvasive and highly selective nanokit for intracellular glucose monitoring via two-step recognition. The liposome-based nanokit coencapsulated the aptamer-functionalized gold nanoparticles (AuNPs) and the Shinkai's receptor together. When the proposed nanokit was transfected into living cells, the Shinkai's receptor could recognize glucose first and then changed its conformation to endow aptamers with binding and sensing properties which were not readily accessible otherwise. Then, the binary complexes formed by the intracellular glucose and the Shinkai's receptor can in situ displace the complementary oligonucleotide of the aptamer on the surface of AuNPs. The fluorophore-labeled aptamer was away from the AuNPs, and the fluorescent state switched from "off" to "on". Through the secondary identification of aptamer, the selectivity of the Shinkai's receptor could be greatly improved while the intracellular glucose level was assessed by fluorescence signal recovery of aptamer. In the follow-up application, the approach exhibits excellent selectivity and is noninvasive for intracellular glucose monitoring under normoxia and hypoxia. To the best of our knowledge, this is the first time that the advantages of organic receptors and nucleic acids have been combined and highly selective monitoring of intracellular glucose has been realized via two-step recognition. We expect it to open up new possibilities to integrate devices for diagnosis of various metabolic diseases and insulin delivery.

It is of scientific significance to explore the intricate relationship between two crucial gasotransmitters nitric oxide (NO) and hydrogen sulfide (H

Inner filter effect (IFE), a well-known phenomenon of fluorescence quenching resulting from absorption of the excitation or emission light of luminescent species by absorbent, has been used as a smart approach to design fluorescent sensors, which are characterized by the simplicity and flexibility with high sensitivity. However, further application of IFE-based sensors in complex environment is hampered by the insufficient IFE efficiency and low sensitivity resulting from interference of the external environment. In this paper, we report that IFE occurring on a solid substrate surface would solve this problem. As a proof of concept, a fluorescent sensor for intracellular biothiols has been developed on the basis of the absorption of a newly designed thiols-specific chromogenic probe (CP) coupled with the use of a thiols-independent fluorophore, rhodamine 6G (R6G), operative on the IFE on graphene oxide (GO). To construct an efficient IFE system, R6G was covalently attached to GO, and the CP molecules were adsorbed on the surface of R6G-GO via π-π stacking interaction. The reaction of thiols with CP on R6G-GO decreases the absorption of CP, resulting in the increase of the intensity of R6G fluorescence. The results showed that the IFE efficiency, sensitivity, and dynamic response time of R6G-GO/CP for biothiols could be significantly improved compared with R6G/CP, and furthermore, R6G-GO/CP functioned under complex system and could be used for assaying biothiols in living cells and in human serum samples. This new strategy would be general to explore the development of more effective IFE-based sensors for other analytes of interest.

Two-photon excitation (TPE) with near-infrared (NIR) photons as the excitation source have the unique properties of lower tissue autofluorescence and self-absorption, reduced photodamage and photobleaching, higher spatial resolution, and deeper penetration depth (500 μm). Carbon nanomaterials, for example, graphene oxide (GO), have the advantages of good biocompatibility, efficient transporters into cells, protecting the carried DNA or peptides from enzymatic cleavage, and super fluorescence quenching efficiency. By combination of the nanostructured carbon materials with the TPE technique, herein we have designed an aptamer-two-photon dye (TPdye)/GO TPE fluorescent nanosensing conjugate for molecular probing in biological fluids, living cells, and zebrafish. This approach takes advantage of the exceptional quenching capability of GO for the proximate TP dyes and the higher affinity of single-stranded DNA on GO than the aptamer-target complex. Successful in vitro and in vivo detection of ATP was demonstrated with this sensing strategy. Our results reveal that the GO/Aptamer-TPdye system not only is a robust, sensitive, and selective sensor for quantitative detection of ATP in the complex biological environment but also can be efficiently delivered into live cells or tissues and act as a "signal-on" in vivo sensor for specific, high-contrast imaging of target biomolecules. Our design provides a methodology model scheme for development of future carbon nanomaterial-based two-photon fluorescent probes for in vitro or in vivo determination of biological or biologically relevant species.

Hypoxia is considered to contribute to pathophysiology in various cells and tissues, and a clear understanding about the relationship between hypoxia and intracellular acidification will help to elucidate the complex mechanism of glycolysis under hypoxia. However, current studies are mainly focused on overexpression of intracellular reductases accelerated by hypoxia, and the investigations focusing on the relationship between hypoxic degree and intracellular acidification remain to be explored. For this vacuity, we report herein a new activatable nanoprobe for sensing pH change under different degrees of hypoxia by surface-enhanced Raman spectroscopy (SERS). The monitoring was based on the SERS spectra changes of 4-nitrothiophenol (4-NTP)-functionalized gold nanorods (AuNR@4-NTP) resulting from the nitroreductase (NTR)-triggered reduction under hypoxic conditions while the as-generated 4-aminothiophenol (4-ATP) is a pH-sensitive molecule. This unique property can ensure the SERS monitoring of intracellular acidification in living cells and tissues under hypoxic conditions. Dynamic pH analysis indicated that the pH decreased from 7.1 to 6.5 as a function of different degrees of hypoxia (from 15 to 1%) due to excessive glycolytic activity triggered by hypoxia. Given the known advantages of SERS sensing, these findings hold promise in studies of pathophysiological pathways involving hypoxia.

Fluorescence anisotropy (FA) is a reliable, sensitive, and robust assay approach for determination of many biological targets. However, it is generally not applicable for the assay of small molecules because their molecular masses are relatively too small to produce observable FA value changes. To address this issue, we report herein the development of a FA signal amplification strategy by employing graphene oxide (GO) as the signal amplifier. Because of the extraordinarily larger volume of GO, the fluorophore exhibits very high polarization when bound to GO. Conversely, low polarization is observed when the fluorophore is dissociated from the GO. As proof-of-principle, the approach was applied to FA detection of adenosine triphosphate (ATP) with a fluorescent aptamer. The aptamer exhibits very high polarization when bound to GO, while the FA is greatly reduced when the aptamer complexes with ATP, which exhibits a maximum signal change of 0.316 and a low detection limit of 100 nM ATP in buffer solution. Successful application of this strategy has been demonstrated that it can be constructed either in a "signal-off" or in a "signal-on" detection scheme. Moreover, because FA is less affected by environmental interferences, FA measurements could be conveniently used to directly detect as low as 1.0 μM adenosine triphosphate (ATP) in human serum. The universality of the approach could be achieved to detect an array of biological analytes when complemented with the use of functional DNA structures.

It is well-known that cyanide ion (CN–) is a hypertoxic anion, which can cause adverse effects in both the environment and living beings; thus, it is highly desirable to develop strategies for detecting CN–, especially in water and food. However, due to the short half-life of free cyanide, long analysis time and/or interference from other competitive ions are general challenges for accurate monitoring of CN–. In this work, through the investigation on the sequence-dependent optical interaction of DNA-CuNPs with the fluorophore (e.g., EBMVC-B), we found, for the first time, that DNA-CuNPs were an ideal alternative as fluorescence quencher in constructing a sensor which could be illuminated by CN– based on an Elsner-like reaction and that the signal switching was dependent on poly(AT/TA) dsDNA sequence. By virtue of CuNPs’ small size and its high chemical reactivity with cyanide, the lighting of fluorescence was ultrarapid and similar to the hairtrigger “turn-on” of a lamp, which is significant for accurate...

The development of nanoprobes suitable for two-photon microscopy techniques is highly desirable for mapping biological species in living systems. However, at the current stage, the nanoprobes are restricted to single-color fluorescence changes, making it unsuitable for quantitative detection. To circumvent this problem, we report here a rational design of a dual-emission and two-photon (TP) graphene quantum dot (GQD(420)) probe for imaging of hydrogen peroxide (H2O2). For specific recognition of H2O2 and lighting the fluorescence of TPGQD(420), a boronate ester-functionalized merocyanine (BMC) fluorophore was used as both target-activated trigger and the dual-emission fluorescence modulator. Upon two-photon excitation at 740 nm, TPGQD(420)-BMC displays a green-to-blue resolved emission band in response to H2O2 with an emission shift of 110 nm, and the H2O2 can be determined from 0.2 to 40 μM with a detection limit of 0.05 μM. Moreover, the fluorescence response of the TPGQD(420)-BMC toward H2O2 is rapid and extremely specific. The feasibility of the proposed method is demonstrated by two-photon ratiometrically mapping the production of endogenous H2O2 in living cells as well as in deep tissues of murine mode at 0-600 μm. To the best of our knowledge, this is the first paradigm to rationally design a dual-emission and two-photon nanoprobe via fluorescence modulation of GQDs with switchable molecules, which will extend new possibility to design powerful molecular tools for in vivo bioimaging applications.

The levels of circulating tumor DNA (ctDNA) in the peripheral blood have been associated with tumor burden and malignant progression. However, ultrasensitive detection of ctDNA in blood remains to be explored. Herein, we have developed a new approach, employing DNA-mediated surface-enhanced Raman scattering (SERS) of single-walled carbon nanotubes (SWNTs), that allows ultrasensitive detection of a broad range of ctDNAs in human blood. Combined with the efficient ctDNA recognition capacity of our designed triple-helix molecular switch and RNase HII enzyme-assisted amplification, the T-rich DNA-mediated SERS enhancement of SWNTs could read out a content of KRAS G12DM as low as 0.3 fM, with a detection of 5.0 μL of sample volume, which has potential for point-of-care testing in clinical analysis.

Complementary electrostatic interaction between the zwitterionic merocyanine and dipolar molecules has emerged as a common strategy for reversibly structural conversion of spiropyrans. Herein, we report a concept-new approach for thermal switching of a spiropyran that is based on simultaneous nucleophilic-substitution reaction and electrostatic interaction. The nucleophilic-substitution at spiro-carbon atom of a spiropyran is promoted due to electron-deficient interaction induced by 6- and 8-nitro groups, which is responsible for the isomerization of the spiropyran by interacting with thiol-containing amino acids. Further, the electrostatic interaction between the zwitterionic merocyanine and the amino acids would accelerate the structural conversion. As proof-of-principle, we outline the route to glutathione (GSH)-induced ring-opening of 6,8-dinitro-1',3',3'-trimethylspiro [2H-1-benzopyran-2,2'-indoline] (1) and its application for rapid and sensitive colorimetric detection of GSH. In ethanol-water (1:99, v/v) solution at pH 8.0, the free 1 exhibited slight-yellow color, but the color changed clearly from slight-yellow to orange-yellow when GSH was introduced into the solution. Ring-opening rate of 1 upon accession of GSH in the dark is 0.45 s(-1), which is 4 orders of magnitude faster in comparison with the rate of the spontaneous thermal isomerization. The absorbance enhancement of 1 at 480 nm was in proportion to the GSH concentration of 2.5 × 10(-8)-5.0 × 10(-6) M with a detection limit of 1.0 × 10(-8) M. Furthermore, due to the specific chemical reaction between the probe and target, color change of 1 is highly selective for thiol-containing amino acids; interferences from other biologically active amino acids or anions are minimal.

Ultrasensitive fluorescent analysis or monitoring of significant molecules in complex samples is important for many biological studies, clinical diagnosis, and forensic investigations, the major obstacle for which is the background signals from ubiquitous endogenous fluorescent components of the environments. Herein, a room-temperature phosphorescence (RTP)-based molecular beacon (MB), employing a Eu(3+) complex of chlorosulfonylated tetradentate β-diketone (L) and the quencher BHQ-2, was engineered for highly sensitive detection of DNA sequences in biological fluids. Complexation of Eu(3+) with the ligand L formed a strongly luminescent complex EuL(2). But when EuL(2) and BHQ-2 were labeled to two ends of a DNA molecule with hairpin structure, the luminescence of EuL(2) was quenched by BHQ-2 due to the stem-closed conformation of the beacon. Due to very low background luminescence from the probe molecule,200-fold signal enhancement was achieved when nanomolar target sequence was introduced. This sensitivity is about 20-fold higher than the level achieved with conventional fluorescence-based molecular beacons. Furthermore, because the Eu(3+) complex has a much longer luminescence lifetime (≈0.8 ms) than that of the background (10 ns), RTP measurements were used to directly detect as low as 500 pM DNA in cell media quantitatively without any sample pretreatment.

We report here a carbon nanotube-based approach for label-free and time-resolved luminescent assay of lysozyme (LYS) by engineering an antilysozyme aptamer and luminescent europium(III) (Eu(3+)) complex. The sensing mechanism of the approach is based on the exceptional quenching capability of carbon nanotubes for the proximate luminescent Eu(3+) complex and different propensities of single-stranded DNA and the DNA/protein complex to adsorb on carbon nanotubes. The luminescence of a mixture of chlorosulfonylated tetradentate β-diketone-Eu(3+) and the antilysozyme aptamer was efficiently quenched by single-walled carbon nanotubes (SWNTs) unless the aptamer interacted with LYS. Due to the highly specific recognition ability of the aptamer for the target and the powerful quenching property of SWNTs for luminescence regents, this proposed approach has a good selectivity and high sensitivity for LYS. In the optimum conditions described,700-fold signal enhancement was achieved for micromolar LYS, and a limit of detection as low as 0.9 nM was obtained, which is about 60-fold lower than those of commonly used fluorescent aptamer sensors. Moreover, due to the much longer lifetime of the Eu(3+) luminescence than those of the ubiquitous endogenous fluorescent components, the time-resolved luminescence technique could be conveniently used for application in complicated biological samples. LYS concentrations in human urine were thus detected using time-resolved luminescence measurement with satisfactory recoveries of 95-98%.

Conformationally constraint nucleic acid probes were usually designed by forming an intramolecular duplex based on Watson-Crick hydrogen bonds. The disadvantages of these approaches are the inflexibility and instability in complex environment of the Watson-Crick-based duplex. We report that this hydrogen bonding pattern can be replaced by metal-ligation between specific metal ions and the natural bases. To demonstrate the feasibility of this principle, two linear oligonucleotides and silver ions were examined as models for DNA hybridization assay and adenosine triphosphate detection. The both nucleic acids contain target binding sequences in the middle and cytosine (C)-rich sequences at the lateral portions. The strong interaction between Ag(+) ions and cytosines forms stable C-Ag(+)-C structures, which promises the oligonucleotides to form conformationally constraint formations. In the presence of its target, interaction between the loop sequences and the target unfolds the C-Ag(+)-C structures, and the corresponding probes unfolding can be detected by a change in their fluorescence emission. We discuss the thermodynamic and kinetic opportunities that are provided by using Ag(+) ion complexes instead of traditional Watson-Crick-based duplex. In particular, the intrinsic feature of the metal-ligation motif facilitates the design of functional nucleic acids probes by independently varying the concentration of Ag(+) ions in the medium.

In recent decades, numerous spiropyran derivatives have been designed and utilized for optical sensing of metal ions. However, there is still less research on spiropyran-based anion sensors. In this work, a new spiropyran compound (L) appended with a pendant bis(2-pyridylmethyl)amine was synthesized and used in fluorescent sensing of pyrophosphate ion (PP(i)) in aqueous solution. The molecular recognition and signal transduction are based on the cooperative ligation interactions and the ligation-induced structural conversion of the spiropyran, which leads to a significant change in the photophysical property of the spiropyran. In an ethanol/water solution (30:70, v/v) at pH 7.4, ligation of L with Zn(2+) causes an intense fluorescence emission at 620 nm at the expense of the original fluorescence at 560 nm. Once PP(i) was introduced, interaction between PP(i) and the L-Zn(2+) complex leads to full quenching of the 620 nm band emission which was concomitant with recovery of the 560 nm band emission, and the fluorescence intensity ratio, F(560)/F(620), is proportional to the PP(i) concentration. Under the optimum condition, the L-Zn(2+) complex responds to PP(i) over a dynamic range of 1.0 x 10(-6) to 5.0 x 10(-4) M, with a detection limit of 4.0 x 10(-7) M. The fluorescence response is highly selective for PP(i) over other biologically related substrates, especially the structurally similar anions, such as phosphate and adenosine triphosphate. The mechanism of interaction among L, Zn(2+), and PP(i) was primarily studied by (1)H NMR, (31)P NMR, and HRMS. To demonstrate the analytical application of this approach, the PP(i) concentration in human urine was determined. It was on the order of 3.18 x 10(-5) M, and the mean value for urinary PP(i) excretion by three healthy subjects was 62.4 micromol/24 h.

To construct efficient oligonucleotide probes, specific nucleic acid is designed as a conformationally constrained form based on the formation of a Watson-Crick-based duplex. However, instability of Watson-Crick hydrogen bonds in a complex biological environment usually leads to high background signal from the probe itself and false positive signal caused by nonspecific binding. To solve this problem, we propose a way to restrict the labeled-dyes in a hydrophobic cavity of cyclodextrin. This bounding, which acts like extra base pairs to form the Watson-Crick duplex, achieves variation of level of space proximity of the two labels and thus the degree of conformational constraint. To demonstrate the feasibility of the design, a stem-containing oligonucleotide probe (P1) for DNA hybridization assay and a stemless one (P2) for protein detection were examined as models. Both oligonucleotides were doubly labeled with pyrene at the 5'- and 3'- ends, respectively. It is the cyclodextrin/pyrene inclusion interaction that allows modulating the degree of conformational constraints of P1 and P2 and thus their background signals and selectivity. Under the optimal conditions, the ratio of signal-to-background of P1/gamma-CD induced by 1.0 equiv target DNA is near 174, which is 4-fold higher than that in the absence of gamma-CD. In addition, the usage of gamma-CD shifts the melting temperature of P1 from 57 to 68 degrees C, which is reasonable for improving target-binding selectivity. This approach is simple in design, avoiding any variation of the stem's length and sequences. Furthermore, the strategy is generalizable which is suited for not only the stem-containing probe but also the linear probe with comparable sensitivity and selectivity to conventional structured DNA probes.

A highly sensitive and specific colorimetry-based rolling circle amplification (RCA) assay method for single-nucleotide polymorphism genotyping has been developed. A circular template is generated by ligation upon the recognition of a point mutation on DNA targets. An RCA amplification is then initiated using the circular template in the presence of Phi29 polymerase. The RCA product can be digested by a restricting endonuclease, and the cleaved DNA fragments can mediate the aggregation of gold nanoparticle-tagged DNA probes. This causes a colorimetric change of the solution as the indicator of the mutation occurrence, which can be detected using UV-vis spectroscopy or viewed by naked eyes. On the basis of the high amplification efficiency of Phi29 polymerase, a mutated target of approximately 70 fM can be detected in this assay. In addition, the protection of the circle template using phosphorothioated nucleotides allows the digestion reaction to be performed simultaneously in RCA. Moreover, DNA ligase offers high fidelity in distinguishing the mismatched bases at the ligation site, resulting in positive detection of mutant targets even when the ratio of the wild-type to the mutant is 10,000:1. The developed RCA-based colorimetric detection scheme was demonstrated for SNP typing of beta-thalassemia gene at position -28 in genomic DNA.

A new strategy for molecular beacon binding readout is proposed by using separation of the molecular recognition element and signal reporter. The signal transduction of the target binding event is based on displacing interaction between the target DNA and a competitor, the signal transducer. The target-free capture DNA is first interacted with the competitor, forming an assembled complex. In the presence of a target DNA that the affinity is stronger than that of the competitor, hybridization between capture DNA and the target disassembles the assembled complex and releases the free competitor to change the readout of the signal reporter. To demonstrate the feasibility of the design, a thymine-rich oligonucleotide was examined as a model system. Hg2+ was selected as the competitor, and mercaptoacetic acid-coated CdTe/ZnS quantum dots served as the fluorescent reporter. Selective binding of Hg2+ between the two thymine bases of the capture DNA forms a hairpin-structure. Hybridization between the capture DNA and target DNA destroys the hairpin-structure, releasing Hg2+ ions to quench the quantum dots fluorescence. Under the optimal conditions, fluorescence intensity of the quantum dots against the concentration of perfect cDNA was linear over the concentration range of 0.1-1.6 microM, with a limit of detection of 25 nM. This new assay method is simple in design, avoiding any oligonucleotide labeling. Furthermore, this strategy is generalizable since any target binding can in principle release the signal transducer and be detected with separated signal reporter.

In this paper, we report the assembly of single-walled carbon nanotubes (SWNTs) and single-stranded DNA to develop a new class of fluorescent biosensors which are able to probe and recognize biomolecular interactions in a homogeneous format. This novel sensing platform consists of a structure formed by the interaction of SWNTs and dye-labeled DNA oligonucleotides such that the proximity of the nanotube to the dye effectively quenches the fluorescence in the absence of a target. Conversely, and very importantly, the competitive binding of a target DNA or protein with SWNTs for the oligonucleotide results in the restoration of fluorescence signal in increments relative to the fluorescence without a target. This signaling mechanism makes it possible to detect the target by fluorescence spectroscopy. In the present study, the schemes for such fluorescence changes were examined by fluorescence anisotropy and fluorescence intensity measurements for DNA hybridization and aptamer-protein interaction studies.

Intracellular pH is an important parameter associated with cellular behaviors and pathological conditions. Quantitative sensing pH and monitoring its changes by near-infrared (NIR) fluorescence imaging with high resolution in living systems are essential but challenging due to the lack of effective probes. To achieve good adaptability, in this study, a class of resolution-tunable ratiometric NIR fluorescent probes, which possess a stable NIR hemicyanine skeleton bearing different substituents, are rationally designed and synthesized, enabling detection through noninvasive optical imaging of organisms. Based on the protonation/deprotonation of the hydroxy group, a marked NIR emission shift provides a ratio signal in response to pH. Meanwhile, two states exhibit good photostability, sensitivity and reversibility, conducive to longtime monitoring of persistent pH changes without disturbance of other biological active species. Among the series, NIR-Ratio-BTZ modified with an electron-withdrawing substituent of benzothiazole exhibited the largest emission shift of about 76 nm from 672 to 748 nm with the pH environment changing from acidic to basic, which could be considered as a good candidate for high resolution pH imaging in live cells, tissues and organisms. Moreover, NIR-Ratio-BTZ has an ideal pK(a) value (pK(a) ≈ 7.2) for monitoring the minor fluctuations of physiological pH near neutrality. The ratiometric fluorescence measurement is beneficial to ensure the accuracy of quantitative measuring pH changes, as well as the real-time monitoring pH-related physiological effects both in living cells and living mice. The results demonstrate that NIR-Ratio-BTZ is a highly sensitive ratiometric pH probe in vivo, giving it potential for biological applications.

Although surface-enhanced Raman spectroscopy (SERS) has been featured by high sensitivity, additional signal enhancement is still necessary for trace amount of biomolecules detection. In this paper, a SERS amplified approach, featuring "ions-mediated cascade amplification (IMCA)", was proposed by utilizing the dissolved silver ions (Ag(+)) from silver nanoparticles (AgNPs). We found that using Ag(+) as linkage agent can effectively control the gaps between neighboring 4-aminobenzenethiol (4-ABT) encoded gold nanoparticles (AuNPs@4-ABT) to form "hot spots" and thus produce SERS signal output, in which the SERS intensity was proportional to the concentration of Ag(+). Inspired by this finding, the IMCA was utilized for ultrasensitive detection of single nucleotide polymorphism in human mitochondrial DNA (16189T → C). Combining with the DNA ligase reaction, each target DNA binding event could successfully cause one AgNP introduction. By detecting the dissolved Ag(+) from AgNPs using IMCA, low to 3.0 × 10(-5) fm/μL targeted DNA can be detected, which corresponds to extractions from 200 nL cell suspension containing carcinoma pancreatic β-cell lines from diabetes patients. This IMCA approach is expected to be a universal strategy for ultrasensitive detection of analytes and supply valuable information for biomedical research and clinical early diagnosis.

Methylmercury (CH3Hg(+)), the common organic source of mercury, is well-known as one of the most toxic compounds that is more toxic than inorganic or elemental mercury. In seabeds, the deposited Hg(2+) ions are converted into CH3Hg(+) by bacteria, where they are subsequently consumed and bioaccumulated in the tissue of fish, and finally, to enter the human diet, causing severe health problems. Therefore, sensitive and selective detection of bioaccumulation of CH3Hg(+) in fish samples is desirable. However, selective assay of CH3Hg(+) in the mercury-containing samples has been seriously hampered by the difficulty to distinguish CH3Hg(+) from ionic mercury. We report here that metal amalgamation, a natural phenomenon occurring between mercury atoms and certain metal atoms, combining with DNA-protected silver nanoparticles, can be used to detect CH3Hg(+) with high sensitivity and superior selectivity over Hg(2+) and other heavy metals. In our proposed approach, discrimination between CH3Hg(+) and Hg(2+) ions was realized by forming Ag/Hg amalgam with a CH3Hg(+)-specific scaffold. We have found that Ag/Hg amalgam can be formed on a CH3Hg(+)-specific DNA template between silver atoms and mercury atoms but cannot between silver atoms and CH3Hg(+). With a dye-labeled DNA strand, the sensor can detect CH3Hg(+) down to the picomolar level, which is125-fold sensitive over Hg(2+). Moreover, the presence of 50-fold Hg(2+) and 10(6)-fold other metal ions do not interfere with the CH3Hg(+) detection. The results shown herein have important implications for the fast, easy, and selective detection and monitoring of CH3Hg(+) in environmental and biological samples.

Levels of lysosomal copper are tightly regulated in the human body. However, few methods for monitoring dynamic changes in copper pools are available, thus limiting the ability to diagnostically assess the influence of copper accumulation on health status. We herein report the development of a dual target and location-activated rhodamine-spiropyran probe, termed Rhod-SP, activated by the presence of lysosomal Cu(2+). Rhod-SP contains a proton recognition unit of spiropyran, which provides molecular switching capability, and a latent rhodamine fluorophore for signal transduction. Upon activation by lysosomal acidic pH, Rhod-SP binds with Cu(2+) by spiropyran-based proton activation, promoting, in turn, rhodamine ring opening, which shows a "switched on" fluorescence signal. However, to protect Rhod-SP from degradation and interference by the physiological environment, it is engineered on mesoporous silica nanoparticles (MSNs), and the surface of Rhod-SP@MSNs is further anchored with β-cyclodextrin (β-CD) to enhance the solubility and bioavailability of Rhod-SP@MSN-CD. Next, to enhance cell specificity, a guiding unit of c(RGDyK) peptide conjugated adamantane (Ad-RGD) as prototypical system, is incorporated on the surface of Rhod-SP@MSN-CD to target integrin αvβ3 and αvβ5 overexpressed on cancer cells. Fluorescence imaging showed that both Rhod-SP@MSN-CD and Rhod-SP@MSN-CD-RGD were suitable for visualizing exogenous and endogenous Cu(2+) in lysosomes of living cells. This strategy addresses some common challenges of chemical probes in biosensing, such as spatial resolution in cell imaging, the solubility and stability in biological system, and the interference from intracellular species. The newly designed nanoprobe, which allows one to track, on a location-specific basis, and visualize lysosomal Cu(2+), offers a potentially rich opportunity to examine copper physiology in both healthy and diseased states.

A growing body of evidence suggests that hydrogen peroxide (H2O2) plays an active role in the regulation of various physiological processes. Development of sensitive probes for H2O2 is an urgent work. In this study, we proposed a DNA-mediated silver nanoparticle and graphene quantum dot hybrid nanocomposite (AgNP-DNA@GQDs) for sensitive fluorescent detection of H2O2. The sensing mechanism is based on the etching effect of H2O2 to AgNPs and the cleavage of DNA by as-generated hydroxyl radicals (•OH). The formation of AgNP-DNA@GQDs nanocomposite can result in fluorescence quenching of GQDs by AgNPs through the resonance energy transfer. Upon H2O2 addition, the energy transfer between AgNPs and GQDs mediated by DNA was weakened and obvious fluorescence recovery of GQDs could be observed. It is worth noting that the reaction product •OH between H2O2 and AgNPs could cleave the DNA-bridge and result in the disassembly of AgNP-DNA@GQDs to achieve further signal enhancement. With optimal conditions, the approach achieves a low detection limit of 0.10 μM for H2O2. Moreover, this nanocomposite is further extended to the glucose sensing in human urine combining with glucose oxidase (GOx) for the oxidation of glucose and formation of H2O2. The glucose concentrations in human urine are detected with satisfactory recoveries of 94.6-98.8% which holds potential for ultrasensitive quantitative analysis of glucose and supplies valuable information for diabetes mellitus research and clinical diagnosis.

Two-photon excitation (TPE) with near-infrared (NIR) photons as the excitation source has important advantages over conventional one-photon excitation (OPE) in the field of biomedical imaging. β-cyclodextrin polymer (βCDP)-based two-photon absorption (TPA) fluorescent nanomicelle exhibits desirable two-photon-sensitized fluorescence properties, high photostability, high cell-permeability and excellent biocompatibility. By combination of the nanostructured two-photon dye (TPdye)/βCDP nanomicelle with the TPE technique, herein we have designed a TPdye/βCDP nanomicelle-based TPA fluorescent nanoconjugate for enzymatic activity assay in biological fluids, live cells and tissues. This sensing system is composed of a trans-4-[p-(N,N-diethylamino)styryl]-N-methylpyridinium iodide (DEASPI)/βCDP nanomicelle as TPA fluorophore and carrier vehicle for delivery of a specific peptide sequence to live cell through fast endocytosis, and an adamantine (Ad)-GRRRDEVDK-BHQ2 (black hole quencher 2) peptide (denoted as Ad-DEVD-BHQ2) anchored on the DEASPI/βCDP nanomicelle's surface to form TPA DEASPI/βCDP@Ad-DEVD-BHQ2 nanoconjugate by the βCD/Ad host-guest inclusion strategy. Successful in vitro and in vivo enzymatic activities assay of caspase-3 was demonstrated with this sensing strategy. Our results reveal that this DEASPI/βCDP@Ad-DEVD-BHQ2 nanoconjugate not only is a robust, sensitive and selective sensor for quantitative assay of caspase-3 in the complex biological environment but also can be efficiently delivered into live cells as well as tissues and act as a "signal-on" fluorescent biosensor for specific, high-contrast imaging of enzymatic activities. This DEASPI/βCDP@Ad-DEVD-BHQ2 nanoconjugate provides a new opportunity to screen enzyme inhibitors and evaluate the apoptosis-associated disease progression. Moreover, our design also provides a methodology model scheme for development of future TPdye/βCDP nanomicelle-based two-photon fluorescent probes for in vitro or in vivo determination of biological or biologically relevant species.

A new zinc(II) porphyrin conjugate with an appended pyrene subunit has been synthesized and shown to exhibit significant and analytical usefulness for fluorescence sensing toward imidazole derivatives. The molecular recognition was based on the bridging interaction of the imidazole ring of analyte with the zinc(II) center of the porphyrin, while the transduction signal for the recognition process was the pyrene excimer fluorescence. The sensor was constructed and applied for fluorescence assay of histidine in aqueous solution by immobilizing the sensing material in a plasticized PVC membrane. When the membrane was bathed in an alkaline solution void of histidine, zinc(II) porphyrin was present in the monomer form, and pyrene emitted monomer fluorescence at 378 and 397 nm. With the presence of histidine in the sample solution, histidine was extracted into the membrane phase and bridged with the Zn(II) center of the porphyrin, causing the monomer porphyrin to be converted to its dimeric species. Since the formation of porphyrin dimer was accompanied by the enhancement of pyrene excimer emission at 454 nm, the chemical recognition process could be directly translated into a fluorescent signal. With the optode membrane M1 described, histidine in sample solution from 6.76 x 10(-7) to 5.01 x 10(-3) M can be determined. The limit of detection was 1.34 x 10(-7) M. The optical selectivity coefficient obtained for histidine over biologically relevant amino acids and anions met the selectivity requirements for the determination of histidine in biological samples. Serum histidine values obtained by the optode membrane fell in the normal range of the content reported in the literature and were in good agreement with those obtained by HPLC.

A new optical chemical sensor for continuous monitoring of aliphatic aldehydes has been proposed based on the reversible chemical reaction between a new sensing reagent, 3,3',5,5'-tetramethyl-N-(9-anthrylmethyl)benzidine (TMAB), and the analytes. TMAB, containing two receptors and two fluorescent reporters, can perform dual fluorescence responses corresponding to the reactions of hydrogen ion and carbonyl compound. When immobilized in a plasticized poly(vinyl chloride) membrane, TMAB extracts aliphatic aldehydes from aqueous solution into the bulk membrane phase and reacts with the analyte by forming a Schiff base. Since the extraction equilibrium and chemical reaction are accompanied by fluorescence increase of the sensing membrane, the chemical recognition process could be directly translated into an optical signal. At pH 3.20, the sensor exhibits a dynamic detection range from 0.017 to 4.2 mM n-butyraldehyde with a limit of detection of 0.003 mM. The forward response time (t95) of the sensor is 3-5 min, and the reverse response time is 5-7 min. The responses of the sensor toward different kinds of aldehydes and ketones depend on the lipophilicity and the reactivity of the analytes. Since the fluorescence enhancement of the sensing membrane at 296 nm/410 nm is only related to the formation of Schiff base, the measurement of aldehydes is independent of pH.

In the present paper, a new cyclodextrin/porphyrin supramolecular sensitizer for zinc ion has been proposed based on the porphyrin dual fluorescence emission ratio. In aqueous solution, meso-tetraphenylporphyrin shows weak fluorescence, while in the presence of alkylated beta-cyclodextrin, it exhibits significant fluorescence enhancement by forming a cyclodextrin/porphyrin inclusion complex. Furthermore, the formation of a supramolecular complex causes a remarkable increase of the porphyrin metalation rate following the porphyrin fluorescence emission changes at two different emission wavelengths. The fluorescence emission of tetraphenylporphyrin at 656-nm bands decreases while that at 606 nm increases upon zinc ion interaction. Thus, the inclusion complex can behave as a ratiometric fluorescent sensor. Theoretically derivative equations for fluorescent ratiometry have been proposed for the first time. The feasibility of the proposed method is demonstrated by the performance of fluorometric detection of zinc ion. With the optimum conditions described, zinc ion in aqueous solution can be determined from 5.0 x 10(-7) to 2.5 x 10(-4) M. As the porphyrin electronic absorption and fluorescence emission are located in the visible range, and the fluorescence changes upon zinc ion interaction show high selectivity over biologically relevant cations, the inclusion complex could be used for biomedical application.

A plasticized polymer film, poly(vinyl chloride) (PVC) incorporated with a specific porphyrin dimer, is shown to exhibit significant and analytical usefulness for optical response toward histidine. The porphyrin dimer containing a free-base porphyrin and a covalently linked metalloporphyrin is shown to be weakly fluorescent as a result of the photoinduced intramolecular electron transfer from the inner free-base porphyrin in singlet excited state to a low-spin cobalt(II). The fluorescence enhancement of the membrane by histidine is based on favorable extraction of histidine into the bulk organic membrane and complexation with the inner metallopophyrin moiety and inhibiting the electron transfer process. With the optode membrane described, histidine in a sample solution from 0.0045 to 1.53 mM can be determined. The calibration curve of the optode membrane for histidine shows a good correlation with the mathematically derived formalism and, thus, confirms the theoretically expected behavior. The sensor presented exhibits high selectivity toward histidine over several amino acids and common inorganic anions. The optical selectivity coefficients obtained for histidine over other biologically relevant amino acids and anions are shown to meet the selectivity requirements for the monitoring concentration levels of histidine in biological samples. The selective characteristic of the sensor has been discussed in the view of the coordination chemistry of metalloporphyrin.