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Precise on-site monitoring of alkylresorcinols, a vital biomarker, is crucial for verifying whole wheat foods and accurately quantifying the whole wheat content in various consumer and industrial products. Herein, for the first time, we introduce a novel ratiometric fluorescence sensor (CDs@ZIF-8/CdTe@MIP) for ultrasensitive and selective detection of alkylresorcinols. 5-Heneicosylresorcinol (C21:0 AR), the primary alkylresorcinol homologue in whole wheat grains, was selected as the target analyte. This analyte was specifically and selectively recognized by the incorporation of a molecularly imprinted polymer (MIP) layer. Within this nanoreactor, blue-emitting carbon dots embedded in zeolitic imidazolate framework-8 (CDs@ZIF-8) and orange-emitting CdTe quantum dots served as the self-calibration signal and response signal, respectively. Exploiting a photoinduced electron transfer effect between CdTe and C21:0 AR, the established fluorescence sensor exhibited remarkable sensing performance, offering wide linear responses in 0.005-1 μg·mL -1 and 1-80 μg·mL -1 concentration ranges, and achieving a low detection limit of 1.14 ng·mL -1 . The proposed assay effectively detected C21:0 AR in real samples, including 8 whole wheat foods and 19 whole wheat grains, demonstrating good recoveries and relative standard deviation. Furthermore, an intelligent sensing platform was established by integrating CDs@ZIF-8/CdTe@MIP with a smartphone-assisted device, thus validating the feasibility of visual and on-site monitoring of C21:0 AR. Because of its rapid response, portability, cost-effectiveness, superior sensitivity, and high selectivity, the proposed sensor serves as a reliable method for the analysis of C21:0 AR, thus having substantial potential for on-site monitoring of whole wheat foods.

The dynamics of peptide-protein binding and unbinding of a variant of the RNase S system has been investigated. To initiate the process, a photoswitchable azobenzene moiety has been covalently linked to the S-peptide, thereby switching its binding affinity to the S-protein. Transient fluorescence quenching was measured with the help of a time-resolved fluorometer, which has been specifically designed for these experiments and is based on inexpensive light-emitting diodes and laser diodes only. One mutant shows on-off behavior with no specific binding detectable in one of the states of the photoswitch. Unbinding is faster by at least 2 orders of magnitude, compared to that of other variants of the RNase S system. We conclude that unbinding is essentially barrier-less in that case, revealing the intrinsic dynamics of the unbinding event, which occurs on a time scale of a few hundred microseconds in a strongly stretched-exponential manner.

Capillary electrophoresis (CE) is a highly efficient separation method capable of handling small sample volumes (∼pL) and low (∼yoctomole) detection limits and, as such, is ideal for applications that require high sensitivity, such as single-cell analysis (Chen et al. Anal. Chem. 1996 , 68 (4), 690-696; Cohen et al. Annu. Rev. Anal. Chem. 2008 , 1 (1), 165-190; Vickerman et al. ACS Chem. Biol. 2018 , 13 (7), 1741-1751). Low-cost CE instrumentation is quickly expanding, but low-cost, open-source fluorescence detectors with ultrasensitive detection limits are lacking (Vickerman et al. ACS Chem. Biol. 2018 , 13 (7), 1741-1751; Fang et al. Electrophoresis 2016 , 37 (17-18), 2376-2383; Casto et al. Anal. Chem. 2019 , 40 (1), 65-78). Silicon photomultipliers (SiPM) are inexpensive, low-footprint detectors with the potential to fill the role as a detector when cost, size, and customization are important. In this work, we demonstrate the use of a SiPM in CE with zeptomolar detection limits and a dynamic range spanning 5 orders of magnitude, comparable to photomultiplier detectors. The performance of these detectors was measured using a continuous wave excitation laser in an epifluorescence detection configuration. We characterize the performance of the SiPM as a highly sensitive detector by measuring enzyme activity in single cells. This simple, small footprint, and low-cost (<$130) light detection circuit will be beneficial for open-source, portable, and budget-friendly instrumentation requiring high sensitivity.

In this paper, a novel and facile synthetic method of 3-mercaptopropionic acid functionalized copper nanoclusters with aggregation-induced emission (AIE) induced by Cu 2+ (Cu 2+ @MPA-Cu NCs) was developed by a one-pot reaction as a fluorescent probe for the detection of sulfide ion (S 2- ). The prepared Cu 2+ @MPA-Cu NCs behaved as aggregated clusters and had strong pink fluorescence under 365 nm UV light with excellent fluorescence emission at 610 nm. The quantum yield increased from 0.56% to 4.8% before and after Cu 2+ added. The presence of S 2- would strongly bind to Cu 2+ , which caused the structure of the aggregated Cu 2+ @MPA-Cu NCs to be destroyed and then the fluorescence quenched. On the basis of this principle, a fluorescent probe was constructed for the detection of S 2- with a very good linearity in the range 0-600 μM ( R 2 = 0.9843) and a detection limit of 26.3 nM. Finally, the nanohybrids were successfully demonstrated for the application in the selective detection of S 2- in food additives. This study essentially paved a new avenue for effectively developing an easy sensor platform for S 2- measurements in food additives.

The estimation of yeast viability with B- and N-doped carbon dots (BN-CDs) was investigated in this paper. BN-CDs with a fluorescent quantum yield of 65.47% were prepared by a one-step hydrothermal method. The size distribution of BN-CDs was relatively narrow, with the majority falling within 7.5-8.5 nm, and they were mainly composed of carbon, oxygen, nitrogen, and boron. BN-CDs were shown to have strong and stable fluorescence. They exhibited excitation-independent photoluminescence property, which could avoid the autofluorescence and limitation of the excitation source. Dead and live yeast cells were distinguished well by BN-CD staining in a short time, and there was no strict requirement for light protection. The application of BN-CDs in beer brewing can solve the problem of estimation of yeast viability.

Cellulose paper has strong potential as an analytical platform owing to its unique characteristics. In the present study, we investigated a procedure for functionalizing the surface of cellulose paper by dip-coating a mixture of a functional polymer and a perfluoroalkylated surfactant (surfactant 1). The functional polymer comprised a mixture of methyl methacrylate and poly(ethylene glycol) methacrylate monomers. The monomer ratio in the functional polymer affected the hydrophilicity and water absorbance of the cellulose paper after dip-coating. Furthermore, the presence of surfactant 1 during dip-coating promoted the surface segregation of poly(ethylene glycol) (PEG) moieties in the polymer, which enhanced the hydrophilicity, prevented nonspecific protein adsorption, and maintained the water absorbance of the dip-coated cellulose paper. Dip-coating with another functional polymer containing biotin groups produced a cellulose paper with a biotin-decorated surface in a one-step procedure. The displayed biotin groups immobilized avidin on the surface, and the PEG moieties in the polymer prevented nonspecific protein adsorption. We then immobilized a thrombin-binding DNA aptamer on the avidin-immobilized cellulose paper to prepare a paper-based analytical device. It is possible to visualize thrombin in model solutions and serum using the paper-based analytical device.

A digital fluorescence detector (DFD), a handheld fluorescence detection device, can convert the fluorescence signal of samples into the corresponding fluorescer concentration. Herein, by adopting a DFD as the readout, a novel intelligent platform was developed based on a ratiometric paper-based device (RPD) for multiple aminoglycoside detection. There are five layers and four parallel channels contained in the designed RPD, functioning as reagent storage, fluidic path control and signal processing, respectively. The rationale of this design lies in the fact that aptamer/graphitic carbon nitride nanosheet (Apt/g-C 3 N 4 NS) modified layers can catalyze o -phenylenediamine to fluorescent 2,3-diaminophenazine (DAP) in the presence of H 2 O 2 . When Apt was removed from nanosheets via the Apt-target reaction, the peroxidase-like activity would be decreased, thus decreasing the production of DAP. All the changes of the fluorescence DAP signal can be read out using a portable DFD. Based on the DFD signal change related to the concentration of the target, a quantitative reaction platform was established. Furthermore, the sample flow and Apt-target reaction time can be reasonably regulated using the H 2 O 2 -cleavable hydrophobic compound modified layer placed between the target recognition region and detection region. Then, the practicality of this platform was verified through realizing sensitive analysis of streptomycin, tobramycin, and kanamycin simultaneously. Overall, with merits including portability and ease of operation, the platform shows great potential in on-site simultaneous detection of multiple targets, especially in resource-limited settings.

In all eukaryotic cells, modifications of proteins by polymers of ubiquitin (polyUb) are signals used in diverse biological processes. To better understand how polyUb signals are read and promote their different functions, quantitative measurements of their interactions with receptor proteins are needed. However, affinities and selectivities of different forms of polyUb with various receptors have been difficult to determine because the availability of well-defined polyUb chains can be limiting and there is a lack of general, sensitive methods to assay their interactions. We have addressed this challenge by developing a series of fluorescent protein sensors for polyUb; by competition of the sensors against receptor proteins in vitro for limiting amounts of polyUb, receptor·polyUb affinities can be quantified. Due to the high affinities of the polyUb sensors ( K d ∼ 10 -9 M), binding assays using this competition format require much less polyUb (<0.1%) than would be needed in direct titrations of the polyUb ligands. Furthermore, the high sensitivity and large dynamic range of the sensor fluorescence readout allow for precise measurements even for very tight interactions (i.e., nanomolar K d ). Importantly, as demonstrated here with Ub 2 and Ub 3 ligands, the assay does not require labeling of either the receptor protein or the polyUb, and it can be used with polyUb ligands composed of virtually any Ub-Ub linkage type.

Single molecule detection provides detailed information about molecular structures and functions but it generally requires the presence of a fluorescent marker which can interfere with the activity of the target molecule or complicate the sample production. Detecting a single protein with its natural UV autofluorescence is an attractive approach to avoid all the issues related to fluorescence labeling. However, the UV autofluorescence signal from a single protein is generally extremely weak. Here, we use aluminum plasmonics to enhance the tryptophan autofluorescence emission of single proteins in the UV range. Zero-mode waveguide nanoapertures enable the observation of the UV fluorescence of single label-free β-galactosidase proteins with increased brightness, microsecond transit times, and operation at micromolar concentrations. We demonstrate quantitative measurements of the local concentration, diffusion coefficient, and hydrodynamic radius of the label-free protein over a broad range of zero-mode waveguide diameters. Although the plasmonic fluorescence enhancement has generated a tremendous interest in the visible and near-infrared parts of the spectrum, this work pushes further the limits of plasmonic-enhanced single molecule detection into the UV range and constitutes a major step forward in our ability to interrogate single proteins in their native state at physiological concentrations.

Recently, luminescent metal-organic framework (MOF) materials have attracted considerable attention in fluorescence sensing. In this essay, we prepared a new cluster-based Ca II -MOFs {[Ca 1.5 (μ 8 -HL 1 )(DMF) 2 ]·DMF} n ( 1 ) with good water dispersibility, excellent photoluminescence properties (FL quantum yield of 20.37%) and great fluorescence stability. Further, it was employed to design as an "off-on" fluorescence sensor for sensitive detection of l-cysteine. This proposed strategy was that fluorescence of Ca II -MOFs 1 was quenched for providing a low fluorescence background by the introduction of Pb 2+ forming the Ca II -MOFs 1 /Pb 2+ hybrid system. The quenching effect could be ascribed to the static quenching mechanism because of the formation of ground-state complexes and coordination interactions between the free carboxyl of H 4 L 1 ligands of Ca II -MOFs 1 and Pb 2+ . Then, with the addition of l-cysteine into the Ca II -MOFs 1 /Pb 2+ hybrid system, the fluorescence signal was immediately restored. This result was because the Pb 2+ was gradually released from the hybrid system by chelation interactions between the -SH groups of l-cysteine and Pb 2+ . This method received a relative wide linear range varying from 0.05 to 40 μM and a low detection limit of 15 nM for detection of l-cysteine. This proposed strategy was also successfully applied to detect l-cysteine in human serum samples with satisfactory recoveries from 95.9 to 101.5%.

As a marker of malignant tumors, miRNA is closely related to the occurrence and metastasis of tumors. How to achieve rapid and sensitive real-time detection is important for clinical prevention and treatment of cancer. In this study, an intelligent detection platform based on smartphone image processing technology made point-of-care testing a reality. This new smart approach could detect multiple targets simultaneously and sensitively. Hydrogel microparticles of different coding modes (shapes, numbers) were prepared by flow lithography to detect different miRNAs. After sandwich immunoassays, different shapes of hydrogels showed different fluorescence intensities depending on their targets. Images were captured by a smartphone and then analyzed by image recognition processing software installed on the smartphone. The concentration of miRNA was obtained within 10 s. The entire reaction process did not exceed 2 h. This intelligent and portable detection platform for miRNAs was reliable and the limit of detection reached the femtomole level. This work provided a demonstration of intelligent, portable, real-time detection of tumor markers.

The precise cellular function of peroxynitrite (ONOO - ) in biosystems remains elusive, primarily owing to being short of ultrasensitive techniques for monitoring its intracellular distribution. In this work, a novel rhodamine B cyclic 1,2-dimethylhydrazine fluorescent chemodosimeter RDMH-PN for highly specific and ultrasensitive monitoring of basal ONOO - in biosystems was rationally designed. The fluorescence titration experiments demonstrated that RDMH-PN was capable of quantitatively detecting 0-100 nM ONOO - (limit of detection = 0.68 nM). In addition, RDMH-PN has outstanding performances of ultrafast measurement, naked-eye detection, and preeminent selectivity toward ONOO - to accurately detect intracellular basal ONOO - . Finally, it has been confirmed that RDMH-PN could not only map the intracellular basal ONOO - level by inhibition tests but also trace the fluctuations of endogenous and exogenous ONOO - levels with diverse stimulations in live cells and zebrafish.

Designing organic saccharide sensors for use in aqueous solution is a nontrivial endeavor. Incorporation of hydrogen bonding groups on a sensor's receptor unit to target saccharides is an obvious strategy but not one that is likely to ensure analyte-receptor interactions over analyte-solvent or receptor-solvent interactions. Phenylboronic acids are known to reversibly and covalently bind saccharides (diols in general) with highly selective affinity in aqueous solution. Therefore, recent work has sought to design such sensors and understand their mechanism for allowing fluorescence with bound saccharides. In past work, binding orientations of several saccharides were determined to dimethylaminomethylphenylboronic acid (DMPBA) receptors with an anthracene fluorophore; however, the binding orientation of d-fructose to such a sensor could not be determined. In this work, we investigate the potential binding modes by generating 20 possible bidentate and six possible tridentate modes between fructose and DMPBA, a simplified receptor model. Gas phase and implicit solvent geometry optimizations, with a myriad functional/basis set pairs, were carried out to identify the lowest energy bidentate and tridentate binding modes of d-fructose to DMPBA. An interesting hydrogen transfer was observed during selected bidentate gas phase optimizations; this transfer suggests a strong sharing of the hydrogen atom between the boronate hydroxyl and amine nitrogen.

Cancer-associated mRNA (mRNA) is an important biomarker for early diagnosis, prognosis, and prediction of treatment responses. Despite recent developments in fluorescence live cell imaging, reliable detection and quantification of mRNA in living cells still remain challenging due to a complicated intracellular environment. Herein, we present a fluorescent nanosensor for live-cell monitoring of cancer-related mRNAs involved in the canonical Wnt/β-catenin signaling pathway. The nanosensor enables rapid and accurate assessment of gene downregulation efficiency in a dose- and time-dependent manner by measuring quantitative fluorescence signal corresponding to β-catenin or its target mRNA levels in living cells. It is expected that the fluorescent nanosensor will be applicable to high-throughput screening for the efficient drug discovery and insightful understanding of the molecular mechanisms of potential drug candidate for cancer treatment.

Considerable interest in using lanthanide complexes in optics have been well-known persisted for a long time. But such molecular-based edifices have been excluded from practical application because of their poor thermal or photo stabilities. Here a novel europium embedded layered double hydroxide (Mg-Al LDH-Eu) has been established and such an inorganic-organic framework demonstrates improved thermal performance due to hydrolysis and poly condensation of the trimethoxysilyl-unit. In addition, the incorporation of a functional building block such as ethylenediamine triacetic acid can significantly minimize the negative effects of hydroxyl groups. In the presence of tetracycline (Tc), the nanoprobe exhibits an "off-on" change in aqueous solution, and the red luminescence can be excited in the visible light range (405 nm). It provides a very sensitive signal response to Tc with an excellent linear relation in the range of 0.1 μM to 5.0 μM, and the detection limit of this probe is measured to be 7.6 nM. This nanoplatform exhibits low cytotoxicity during in vitro experiments and can be employed for the detection of tetracycline in 293T cells.

Nitrogen- and sulfur-codoped carbon dots (N,S-CDs) with a fluorescence quantum yield of 16.1% and good photoluminescent properties were synthesized by a simple hydrothermal method. Cytotoxicity of the N,S-CDs was evaluated by the MTT assay, and human hepatoma HepG2 cells were chosen as the target. The cell viability was more than 85% after 24 h of incubation when its concentration was up to 300 μg/mL, suggesting low cytotoxicity and good biocompatibility of the N,S-CDs. The fluorescence spectra of the N,S-CDs are excitation-dependent in the excitation-wavelength range of 295-400 nm, and it emits bright blue fluorescence centered at 435 nm. Its selective fluorescence recognition for Hg 2+ ions was found. When Hg 2+ ions were added to the N,S-CDs solution, its bright blue fluorescence was obviously quenched and could be recovered by the addition of sulfide. Accordingly, a new strategy based on N,S-CDs-Hg 2+  system as a highly selective and ultrasensitive "turn off-on" fluorescence sensing for the detection of sulfide was fabricated. The limits of detection (S/N = 3) are 83 nM for Hg 2+ ions and 11 nM for sulfide. Almost no statistically significant interference for Hg 2+ -ion and sulfide detection was observed among possible coexisting substances in the water samples, including 17 common metal ions and 11 anions. This probe was successfully applied for the cellular imaging of Hg 2+ ions in HepG2 cells by fluorescence microscopy.

An imidazolium-derived pyrene aggregation was developed to rapidly identify and quantify different bacteria species. When the nonemissive aggregates bound to the anionic bacteria surface, the sensor disassembled to turn on significant fluorescence. At the same time, ratiometric signals between pyrene monomer and excimer emission were controlled by different interactions with various bacteria surfaces. The resulted different fluorescent emission profiles then were obtained as fingerprints for various bacterial species. By converting emission profiles directly into output signals of two channels, fluorescence increase and ratiometric change, a two-dimensional analysis map was generated for bacteria identification. We demonstrated that our sensor rapidly identified 10 species of bacteria and 14 clinical isolated multidrug-resistant bacteria, and we determined their staining properties (Gram-positive or Gram-negative).

A simple and miniature analytical system was developed to determine Hg and Cd in small amounts of samples by integrating flow injection capillary liquid electrode discharge (CLED) optical emission spectrometry (OES) and microplasma-induced vapor generation (PIVG) atomic fluorescence spectrometry (AFS). With the assistance of the inherent capillary driving force and the force arising from the solution vaporization in the microplasma, the sample solution was automatically transported into the discharge chamber wherein analytes were simultaneously excited to generate their atomic emission lines and converted to their volatile species. Subsequently, the volatile species were further swept into AFS for their further determination. Therefore, the same sample could be successively analyzed by OES and AFS. Owing to the unique independent linear-range and sensitivity of CLED-OES and PIVG-AFS, the developed system not only significantly extended its linear range to 6 orders of magnitude but also remarkably reduced the sample consumption to several microliters. Thus, wide linear-range and ultrasensitive determination of Hg and Cd in limited amounts of samples were accomplished simply by sharing one single capillary liquid electrode discharge source. Under the optimized conditions, limits of detection (LODs) of 10 μg L -1 were obtained for both Hg and Cd when CLED-OES was used as a detector, whereas the LODs for Hg and Cd were improved to 0.03 μg L -1 and 0.04 μg L -1 with AFS detector, respectively. In addition, the extremely wide linear-range of 0.001-100 mg L -1 and 0.001-40 mg L -1 were obtained for Hg and Cd, respectively. The potential application of this method was validated by successfully analyzing three Certified Reference Materials (ZK021-1, GBW(E)090033, and GBW(E)090034) and six human blood samples.

Constructing polycyclic aromatics-based, highly emissive fluorophores with good solubility and tunable aggregated structures and properties is of great importance for film fabrication, solution processing, and relevant functionality studies. Herein, we describe a general strategy to endow conventional organic fluorophores with enhanced solubility and modulated fluorescent properties via their incorporation into coordination-driven self-assembled metallacycles. A widely used fluorophore, pyrene, was decorated with two pyridyl groups to yield functionalized pyrene 4. Mixing 4 with three aromatic dicarboxylates with different lengths and a 90° Pt(II) metal acceptor in a 2:2:4 stoichiometric ratio resulted in the formation of three metallacycles, 1, 2, and 3. The metallacycles display good solubility in polar organic solvents, highly aggregation-dependent fluorescence, and size-dependent emissions at higher concentrations. Moreover, metallacycle 2-based, silica-gel-supported film as fabricated not only is more emissive than the ligand 4-based one but also displays much improved sensing properties for amines in the vapor state, as demonstrated by significantly increased response speed and decreased recovery time. The enhanced solubility, unique fluorescence behavior, and multi-factor modulation character show that coordination-driven self-assembly can be utilized for the development of new fluorophores through simple modification of conventional fluorophores. The fluorophores synthesized this way possess not only complex topological structures but also good modularity and tunability in fluorescence behavior, which are important for grafting multi-stage energy-transfer systems necessary for the development of high-performance sensing materials.

Food fraud can be highly lucrative, and high accuracy authentication of various foodstuffs is becoming essential. Olive oil is one of the most investigated food matrices, due to its high price and low production globally, with recent food fraud examples showing little or no high quality olive oil in the tested oils. Here a simple method using a 405 nm LED flashlight and a smartphone is developed for edible oil authentication. Identification is fingerprinted by intrinsic fluorescent compounds in the oils, such as chlorophylls and polyphenols. This study uses the hue parameter of HSV-colorspace to authenticate 24 different edible oils of 9 different types and 15 different brands. For extra virgin olive oil, all nine samples are well separated from the other oil samples. The rest of the samples were also well type-distinguished by the hue parameter, which is complemented by hue-histogram analysis. This opens up opportunities for low-cost and high-throughput smartphone field-testing of edible oils on all levels of the production and supply chain.

The sensitive simultaneous detection of multiple biomarkers is critical for the early diagnosis of diseases. Electrochemiluminescence (ECL) offers outstanding advantages, e.g., low background, over other optical sensing techniques. However, multiplexed ECL bioassay is hindered not only by the lack of generally available ECL spectrometers but also by the limited number of biocompatible monochromatic ECL luminophores for decades. Herein, we report addressing these issues by re-examination of the recent tabletop spectrofluorometer coupled potentiostat as a high-resolution ECL spectrum acquisition system and using carbon nitrides as monochromatic luminophores. A wavelength-resolved multiplexing ECL biosensor is demonstrated to simultaneously detect CA19-9 and mesothelin, two pancreatic cancer biomarkers, at a single-electrode interface. This work could initiate new opportunities for more general multiplex ECL biosensors with competitive performances.

Plasmon-enhanced fluorescence (PEF)-based analytical technology has recently demonstrated its ability in detecting biomarkers with ultrahigh sensitivity. However, the scope of the PEF-based technology has been hindered by its reliance on flat substrates with relatively low binding kinetics and the limited multiplex detection ability. Herein, we reported a simple yet robust method for the fabrication of plasmonic magnetic microbeads (PMMBs)-based suspension array technology (SAT) with fluorescence enhancement of about 60-fold, improving the detection limit of biomarkers by 2-orders of magnitude toward 100 fM. We also demonstrated the performance of this method for the detection of anti-acidic ribosomal phosphoprotein 0 (anti-P0) autoantibody in sera from systemic lupus erythematosus (SLE) patients. Owing to the high sensitivity and efficient magnet-based sample collection, our method can be employed for detection of ultrasmall volumes of samples (e.g., 2 μL), promising for point-of-care detection. Furthermore, a size-encoded PMMBs-based multiplexed suspension array for simultaneous detection of multiple biomarkers is realized, illustrating the great potential of this technology in high-throughput disease diagnosis applications.

Caspase-3 is a key enzyme executing apoptosis during ontogenesis and homeostasis of multicellular organisms, and is a very important and potential drug target in treatment of apoptosis disturbance. So far, no commercial drugs for caspase-3 are available, and it is urgently necessitated to develop an effective method for caspase-3 activity assay and its inhibitor screening. In this paper, we propose a new method for determination of caspase-3 activity and its inhibition constant by combining single molecule fluorescence correlation spectroscopy (FCS) with a microwell chip. Its principle is based on measurement of the enzyme reaction kinetics and homogeneous detection of the reaction product by FCS. This system can reduce the requirement sample volume to 1 μL level. The caspase-3 substrates are doubly labeled with fluorophore and biotin, the enzyme reaction can be quickly terminated in the presence of streptavidin, and the reaction products can be selectively detected by FCS. We established the model of caspase-3 inhibitor screening by combining the dynamics of enzyme reaction with FCS theory. This new method was successfully used for determination of inhibition constants of certain inhibitors and assay of drug-induced apoptosis. Compared to current methods, this method shows high sensitivity, small reagent dosage and short analysis time. We believe that this method will become an efficient platform for screening of caspase-3 inhibitors and detection of apoptosis.

The development of ultrasensitive and highly selective fluorescence biosensors for diverse analytes is highly desirable but remains a challenge. It is attributable to the scarcity of fluorogens with promising brightness, stability, and nontoxicity, which primarily determine the performance of fluorescence biosensors. Herein, we report the design and preparation of aggregation induced emission (AIE) dots with high brightness, exceptional colloidal stability, ultrasmall size, and functional groups for developing ultrasensitive biosensor through the electrostatic conjugation to biological molecules, and use blemycin (BLM) as the proof-of-concept analyte. The recognition and the subsequent cleavage of the quencher-labeled DNA (Q-DNA) by BLM result in the formation of three-mer quencher-linked oligonucleotide fragments (Q-DNA-1), which significantly decreases the amount of quenchers anchored on AIE dot surfaces and subsequently reduces the fluorescence resonance energy transfer (FRET) effect. As compared to the case in which BLM is absent, remarkable fluorescence enhancement is observed, and is dependent on BLM concentration. Thus, ultrasensitive fluorescence detection of target BLM is realized, with a detection limit down to 3.4 fM, the lowest value reported so far. Moreover, the proposed fluorescence biosensor has also been successfully utilized for detection of BLM spiked in human serum samples. The as-proposed strategy not only significantly improves the selectivity and sensitivity of BLM assay, but also allows the ultrasensitive detection of a variety of bioactive molecules by simply changing the specific target recognition substances, thus providing a versatile fluorescence platform, and showing great potential to be applied in chemo-/bioanalysis and clinical biomedicine.

In vitro biosensors have been an integral component for early diagnosis of cancer in the clinic. Among them, no-wash biosensors, which only depend on the simple mixing of the signal generating probes and the sample solution without additional washing and separation steps, have been found to be particularly attractive. The outstanding advantages of facile, convenient, and rapid response of no-wash biosensors are especially suitable for point-of-care testing (POCT). One fast-growing field of no-wash biosensor design involves the usage of nanomaterials as signal amplification carriers or direct signal generating elements. The analytical capacity of no-wash biosensors with respect to sensitivity or limit of detection, specificity, stability, and multiplexing detection capacity is largely improved because of their large surface area, excellent optical, electrical, catalytic, and magnetic properties. This review provides a comprehensive overview of various nanomaterial-enhanced no-wash biosensing technologies and focuses on the analysis of the underlying mechanism of these technologies applied for the early detection of cancer biomarkers ranging from small molecules to proteins, and even whole cancerous cells. Representative examples are selected to demonstrate the proof-of-concept with promising applications for in vitro diagnostics of cancer. Finally, a brief discussion of common unresolved issues and a perspective outlook on the field are provided.

An all-in-one paper-based analytical device (PAD) was successfully developed for visual fluorescence detection of carcinoembryonic antigen (CEA) on CdTe/CdSe quantum dot (QD)-enzyme-impregnated paper by coupling with a bioresponsive controlled-release system from DNA-gated mesoporous silica nanocontainers (MSNs). The assay was carried out in a centrifuge tube by using glucose-loaded MSNs with a CEA aptamer and a QD-enzyme-paper attached on the lid. Initially, single-strand complementary DNA to a CEA aptamer was covalently conjugated to the aminated MSN, and then glucose (enzyme substrate) molecules were gated into the pore with the help of the aptamer. Glucose oxidase (GOD) and CdTe/CdSe QDs were coimmobilized on paper for the visual fluorescence signal output. Upon target CEA introduction in the detection cell, the analyte specifically reacted with the immobilized aptamer on the MSN to open the pore, thereby resulting in the glucose release. The released glucose was oxidized by the immobilized GOD on paper to produce gluconic acid and hydrogen peroxide, and the latter quenched the fluorescence of CdTe/CdSe QDs, which could be determined by the naked eye on a portable smartphone and a commercial fluorospectrometer. Under optimal conditions, the PAD-based sensing system enabled sensitive discrimination of target CEA against other biomarkers or proteins in a linear range of 0.05-20 ng mL -1 with a limit of detection of 6.7 pg mL -1 (ppt). In addition, our strategy displayed high specificity, good reproducibility, and acceptable accuracy for analyzing human serum specimens with a commercial human CEA ELISA kit. Importantly, this methodology offers promise for simple analysis of biological samples and is suitable for use in the mass production of miniaturized devices, thus opening new opportunities for protein diagnostics and biosecurity.

Key challenges with point-of-care (POC) nucleic acid tests include achieving a low-cost, portable form factor, and stable readout, while also retaining the same robust standards of benchtop lab-based tests. We addressed two crucial aspects of this problem, identifying a chemical additive, hydroxynaphthol blue, that both stabilizes and significantly enhances intercalator-based fluorescence readout of nucleic acid concentration, and developing a cost-effective fiber-optic bundle-based fluorescence microplate reader integrated onto a mobile phone. Using loop-mediated isothermal amplification on lambda DNA we achieve a 69-fold increase in signal above background, 20-fold higher than the gold standard, yielding an overall limit of detection of 25 copies/μL within an hour using our mobile-phone-based platform. Critical for a point-of-care system, we achieve a >60% increase in fluorescence stability as a function of temperature and time, obviating the need for manual baseline correction or secondary calibration dyes. This field-portable and cost-effective mobile-phone-based nucleic acid amplification and readout platform is broadly applicable to other real-time nucleic acid amplification tests by similarly modulating intercalating dye performance and is compatible with any fluorescence-based assay that can be run in a 96-well microplate format, making it especially valuable for POC and resource-limited settings.

We report on a method for performing ultrafast infrared (IR) vibrational spectroscopy using fluorescence detection. Vibrational dynamics on the ground electronic state driven by femtosecond mid-infrared pulses are detected by changes in fluorescence amplitude resulting from modulation of a two-photon visible transition by nuclear motion. We examine a series of coumarin dyes and study the signals as a function of solvent and excitation pulse parameters. The measured signal characterizes the relaxation of vibrational populations and coherences but yields different information than conventional IR transient absorption measurements. These differences result from the manner in which the ground-state dynamics are projected by the two-photon detection step. Extensions of this method can be adapted for a variety of increased-sensitivity IR measurements.

Aqueous sulfides are emerging signaling agents implicated in various pathological and physiological processes. The development of sensitive and selective methods for the sensing of these sulfides is therefore very important. Herein, we report that the as-synthesized 1-oxo-1H-phenalene-2,3-dicarbonitrile (OPD) compound provides promising fluorescent properties and unique reactive properties toward aqueous sulfides. It was found that OPD showed high selectivity and sensitivity toward Na2S over thiols and other inorganic sulfur compounds through a sulfide involved reaction which was confirmed by high-resolution mass spectroscopy (HRMS) and nuclear magnetic resonance (NMR) results. The fluorescence intensity increases linearly with sulfide concentration in the range of 1.0-30 μM with a limit of detection of 52 nM. This novel fluorescent probe was further exploited for the fluorescence imaging sensing of aqueous sulfide in HeLa cells.

Due to their size and difficulty to obtain, cost/effective biological or synthetic receptors (e.g., antibodies or aptamers, respectively), organic toxic compounds (e.g., less than 1 kDa) are generally challenging to detect using simple platforms such as biosensors. This study reports on the synthesis and characterization of a novel multifunctional composite material, magnetic silica beads/graphene quantum dots/molecularly imprinted polypyrrole (mSGP). mSGP is engineered to specifically and effectively capture and signal small molecules due to the synergy among chemical, magnetic, and optical properties combined with molecular imprinting of tributyltin (291 Da), a hazardous compound, selected as a model analyte. Magnetic and selective properties of the mSGP composite can be exploited to capture and preconcentrate the analyte onto its surface, and its photoluminescent graphene quantum dots, which are quenched upon analyte recognition, are used to interrogate the presence of the contaminant. This multifunctional material enables a rapid, simple and sensitive platform for small molecule detection, even in complex mediums such as seawater, without any sample treatment.

In this work, we designed a dual-emission ratiometric fluorescence probe by hybridizing two differently colored quantum dots (QDs), which possess a built-in correction that eliminates the environmental effects and increases sensor accuracy. Red emissive QDs were embedded in the silica nanoparticle as reference while the green emissive QDs were covalently linked to the silica nanoparticle surface to form ratiometric fluorescence probes (RF-QDs). Dopamine (DA) was then conjugated to the surface of RF-QDs via covalent bonding. The ratiometric fluorescence probe functionalized with dopamine (DA) was highly reactive toward tyrosinase (TYR), which can catalyze the oxidization of DA to dopamine quinine and therefore quenched the fluorescence of the green QDs on the surface of ratiometric fluorescence probe. With the addition of different amounts of TYR, the ratiometric fluorescence intensity of the probe continually varied, leading to color changes from yellow-green to red. So the ratiometric fluorescence probe could be utilized for sensitive and selective detection of TYR activity. There was a good linear relationship between the ratiometric fluorescence intensity and TYR concentration in the range of 0.05-5.0 μg mL(-1), with the detection limit of 0.02 μg mL(-1). Significantly, the ratiometric fluorescence probe has been used to fabricate paper-based test strips for visual detection of TYR activity, which validates the potential on-site application.

High-yield preparation of ultrathin two-dimensional (2D) nanosheets is of great importance for the further exploration of their unique properties and promising applications. Herein, for the first time, the high-yield and scalable production of ultrathin 2D ternary chalcogenide nanosheets, including Ta2NiS5 and Ta2NiSe5, in solution is achieved by exfoliating their layered microflakes. The size of resulting Ta2NiS5 and Ta2NiS5 nanosheets ranges from tens of nanometers to few micrometers. Importantly, the production yield of single-layer Ta2NiS5 nanosheets is very high, ca. 86%. As a proof-of-concept application, the single-layer Ta2NiS5 is used as a novel fluorescence sensing platform for the detection of DNA with excellent selectivity and high sensitivity (with detection limit of 50 pM). These solution-processable, high-yield, large-amount ternary chalcogenide nanosheets may also have potential applications in electrocatalysis, supercapacitors, and electronic devices.

A simple and dual chemical reaction-based multimodal assay for dopamine with high sensitivity and selectivity using two types of functionalized gold nanoparticles (FB-AuNPs/NsNHS-AuNPs), i.e. fluorescein modified gold nanoparticles (FB-AuNPs) and Nile blue modified gold nanoparticles (NsNHS-AuNPs), was successfully fabricated. This assay for dopamine presents colorimetric visualization and double channel fluorescence enhancement at 515 and 665 nm. The absorbance and fluorescence changes were linearly proportional to the amounts of dopamine in the range of nanomolar scale (5-100 nM). The detection limits for absorbance and fluorescence were as low as 1.2 nM and 2.9 nM (S/N = 3), respectively. Furthermore, the extent application of this multimodal assay has been successfully demonstrated in human urine samples with high reliability and applicability, showing remarkable promise in diagnostic purposes.

A near-infrared fluorescent dye (aza-bodipy or azaBDPBA) functionalized with boronic acid groups was synthesized for the preparation of optodes to measure glucose in 40-fold diluted whole blood. Boronic acid groups as an electron deficient group on aza-bodipy was reacted with hydrogen peroxide into an electron-rich phenolic group leading to the red-shift of emission wavelength from 682 to 724 nm. The emission in near-infrared region offered a low level of background interference from whole blood. Also, the dual-wavelength emission guaranteed our probe to measure glucose in whole blood accurately after the conversion of glucose into hydrogen peroxide using glucose oxidase. The measuring range of glucose from 0.2 to 200 mM in the buffer was achieved with high selectivity. To facilitate the blood test, the probe was immobilized into thin hydrophobic polymer films to prepare the disposal glucose optode, which could detect glucose in the solution from 60 μM to 100 mM. The concentration of glucose in 40-fold diluted whole blood was determined using our optode and the reference method, respectively. The consistence in the concentration obtained from these two assays revealed that our azaBDPBA-based optodes were promising for the clinic assay of glucose in the whole blood.

A simple and time-saving spectrofluorometric method developed using an azaflavanon-3-ol compound was used for the determination of iron in various food samples. Nitric acid and hydrogen peroxide were used for digestion of samples in a closed microwave system. The method was validated by analyzing two certified reference materials (CRM-SA-C Sandy Soil C and Mixed Polish Herbs INCT-MPH-2). Measurements were carried out using a modified standard addition method. The standard addition graph was linear until 21.6 mg/L in the determination of iron(III). Detection and quantification limits were 0.81 and 2.4 mg/L, respectively. Satisfactory accuracy was obtained for spinach, dill, mint, purslane, rocket, red lentils, dry beans, and two iron medicinal tablets. High recoveries were found for streamwater samples fortified at three different concentrations. The method is simple, time-saving, cost-effective, and suitable for the determination of the iron content of foods.

Fluorescence sensing of specific biological molecules by artificial chemosensors is a versatile technique. In the present work, a switch-on fluorescence sensor for rapid, sensitive, and selective sensing of glutathione (GSH) in food samples was developed. This method was based on the g-CNQDs-Hg(2+) system, in which the initial fluorescence from g-CNQDs was quenched by Hg(2+) with an electron transfer process. In the presence of GSH, the fluorescence sensor was switched to the "on" state, which was attributed to a competitive affinity of Hg(2+) to GSH and the functional groups on the surface of g-CNQDs. Under the optimal conditions, the limit of detection (LOD) of 37 nM for GSH was achieved with a wide range of 0.16-16 μM. The repeatability was better than 5.3% for GSH in both standard and food samples (n = 3). Finally, this fluorescence sensor was successfully employed for the determination of GSH in various kinds of food samples with excellent recoveries. Furthermore, this application may pave a new way for fluorescence sensing of other substances in food samples.

Dissolved organic matter (DOM) is known to form strong complexes with heavy metals and thus governs the distribution, toxicity, bioavailability, and ultimate fate of heavy metals in the environment. The relevant aspects of metal-organic interactions remain unclear because the metal binding functionalities in DOM are substantially nonuniform and the availability of the models is limited. In this work, two-dimensional correlation spectroscopy (2DCOS) integrated with synchronous fluorescence and infrared absorption spectroscopy was used to explore the binding process of copper to DOM. A series of heterogeneous binding sites in humic acid (HA), a representative DOM, and the subsequent subtle changes of these sites within the molecular interactions were elucidated by the 2DCOS method. The band assignments and the correspondence between the results obtained by two spectral probes (synchronous fluorescence and infrared absorption spectra) were verified by hetero-2DCOS. Our results showed that, during the copper binding process, the carboxyl and polysaccharide groups gave the fastest responses to copper binding. Then fluorescence quenching of fluorescent humic-like moieties occurred with a vibrational change of the related functionalities, i.e., phenolic and aryl carboxylic groups, which further induces the fluorescence quenching of fulvic-like fractions. Finally, small amounts of amide and aliphatic groups participated in the copper binding after the fluorescence of the protein-like fraction decreased. With these promising results, a comprehensive picture of structural changes of HA during the copper binding process was developed, highlighting the superior potential of 2D heterospectral correlation spectroscopy in studying complex interactions in the environment.

A high-sensitivity immunoassay system with surface plasmon field-enhanced fluorescence spectrometry (SPFS) was constructed using a plastic sensor chip and then applied to the detection of total prostate-specific antigen (total PSA) and GalNAcβ1-4GlcNAc-linked prostate-specific antigen (LacdiNAc-PSA) in serum, to discriminate between prostate cancer (PC) and benign prostate hyperplasia (BPH). By using this automated SPFS immunoassay, the detection limit for total PSA in serum was as low as 0.04 pg/mL, and the dynamic range was estimated to be at least five digits. A two-step sandwich SPFS immunoassay for LacdiNAc-PSA was constructed using both the anti-PSA IgG antibody to capture PSA and Wisteria floribunda agglutinin (WFA) for the detection of LacdiNAc. The results of the LacdiNAc-PSA immunoassay with SPFS showed that the assay had a sensitivity of 20.0 pg/mL and permitted the specific distinction between PC and BPH within the PSA gray zone. These results suggested that high-sensitivity automated SPFS immunoassay systems might become a powerful tool for the diagnosis of PC and other diseases.

Understanding surface and interfacial lateral organization in material and biological systems is critical in nearly every field of science. The continued development of tools and techniques viable for elucidation of interfacial and surface information is therefore necessary to address new questions and further current investigations. Sum frequency spectroscopy (SFS) is a label-free, nonlinear optical technique with inherent surface specificity that can yield critical organizational information on interfacial species. Unfortunately, SFS provides no spatial information on a surface; small scale heterogeneities that may exist are averaged over the large areas typically probed. Over the past decade, this has begun to be addressed with the advent of SFS microscopy. Here we detail the construction and function of a total internal reflection (TIR) SFS spectral and confocal fluorescence imaging microscope directly amenable to surface investigations. This instrument combines, for the first time, sample scanning TIR-SFS imaging with confocal fluorescence microscopy.

Several studies have shown high concentrations of polycyclic aromatic hydrocarbons (PAHs) in living spaces and soil adjacent to parking lots sealed with coal-tar-based products. Recent attention has been paid to the presence of seven PAHs in coal-tar samples, namely, benz[a]anthracene, benzo[k]-fluoranthene, benzo[b]fluoranthene, benzo[a]pyrene, chrysene, dibenz[a,h]anthracene, and indeno[1,2,3-cd]pyrene, and their association to significant increases in estimated excess lifetime cancer risk for nearby residents. Herein, we present an analytical approach to screen the presence of five highly toxic, high-molecular weight PAHs (HMW-PAHs) in coal-tar samples. These include dibenzo[a,l]pyrene, dibenzo[a,i]pyrene, dibenzo[a,e]pyrene, dibenzo[a,h]pyrene, and naphtho[2,3-a]pyrene. Their direct analysis, without chromatographic separation, in a reference coal-tar sample is made possible with the combination of excitation-emission matrices (EEMs) and parallel factor analysis (PARAFAC). EEMs are recorded at 4.2 K with the aid of a cryogenic fiber-optic probe and a commercial spectrofluorimeter. The simplicity of the experimental procedure and the excellent analytical figures of merit demonstrate the screening potential of this environmentally friendly approach for the routine analysis of numerous coal-tar samples.

A novel chemical warfare agent sensor based on conjugated polymer dots (CPdots) immobilized on the surface of poly(vinyl alcohol) (PVA)-silica nanofibers was prepared with a dots-on-fibers (DoF) hybrid nanostructure via simple electrospinning and subsequent immobilization processes. We synthesized a polyquinoxaline (PQ)-based CP as a highly emissive sensing probe and employed PVA-silica as a host polymer for the elctrospun fibers. It was demonstrated that the CPdots and amine-functionalized electrospun PVA-silica nanofibers interacted via an electrostatic interaction, which was stable under prolonged mechanical force. Because the CPdots were located on the surface of the nanofibers, the highly emissive properties of the CPdots could be maintained and even enhanced, leading to a sensitive turn-off detection protocol for chemical warfare agents. The prepared fluorescent DoF hybrid was quenched in the presence of a chemical warfare agent simulant, due to the electron transfer between the quinoxaline group in the polymer and the organophosphorous simulant. The detection time was almost instantaneous, and a very low limit of detection was observed (∼1.25 × 10(-6) M) with selectivity over other organophosphorous compounds. The DoF hybrid nanomaterial can be developed as a rapid, practical, portable, and stable chemical warfare agent-detecting system and, moreover, can find further applications in other sensing systems simply by changing the probe dots immobilized on the surface of nanofibers.

Three-photon excitation fluorescence correlation spectroscopy was used to detect oligomerization equilibria of rat liver phosphofructokinase. The fluorescence intensity produced by the three-photon excitation of tryptophan was collected using the DIVER microscope. In this home-built upright microscope, a large area photomultiplier, placed directly below the sample, is used as the detector. The lack of optical elements in the microscope detection path results in a significantly improved detection efficiency in the UV region down to about 300 nm, which encompasses the fluorescence emission from tryptophan. The three-photon excitation autocorrelation decays obtained for phosphofructokinase in the presence of F6P showed the presence of large oligomers. Substitution of F6P with ATP in the buffer medium results in dissociation of the large oligomers, which is reported by the decreased autocorrelation amplitude. The three-photon excitation process was verified from the slope of the log-log plot of intensity against laser power.

The brightness of nanoscale optical materials such as semiconductor nanocrystals is currently limited in high excitation flux applications by inefficient multiexciton fluorescence. We have devised a solution-phase photon correlation measurement that can conveniently and reliably measure the average biexciton-to-exciton quantum yield ratio of an entire sample without user selection bias. This technique can be used to investigate the multiexciton recombination dynamics of a broad scope of synthetically underdeveloped materials, including those with low exciton quantum yields and poor fluorescence stability. Here, we have applied this method to measure weak biexciton fluorescence in samples of visible-emitting InP/ZnS and InAs/ZnS core/shell nanocrystals, and to demonstrate that a rapid CdS shell growth procedure can markedly increase the biexciton fluorescence of CdSe nanocrystals.

We demonstrate the first use of smartphone spectrophotometry for readout of fluorescence-based biological assays. We evaluated the smartphone fluorimeter in the context of a fluorescent molecular beacon (MB) assay for detection of specific nucleic acid sequences in a liquid test sample and compared performance against a conventional laboratory fluorimeter. The capability of distinguishing a one-point mismatch is also demonstrated by detecting single-base mutation in target nucleic acids. Our approach offers a route toward portable biomolecular assays for viral/bacterial pathogens, disease biomarkers, and toxins.

5-Hydroxymethylcytosine (5hmC), a modified form of the DNA base cytosine, is an important epigenetic mark linked to regulation of gene expression in development, and tumorigenesis. We have developed a spectroscopic method for a global quantification of 5hmC in genomic DNA. The assay is performed within a multiwell plate, which allows simultaneous recording of up to 350 samples. Our quantification procedure of 5hmC is direct, simple, and rapid. It relies on a two-step protocol that consists of enzymatic glucosylation of 5hmC with an azide-modified glucose, followed by a "click reaction" with an alkyne-fluorescent tag. The fluorescence intensity recorded from the DNA sample is proportional to its 5hmC content and can be quantified by a simple plate reader measurement. This labeling technique is specific and highly sensitive, allowing detection of 5hmC down to 0.002% of the total nucleotides. Our results reveal significant variations in the 5hmC content obtained from different mouse tissues, in agreement with previously reported data.

Fluorescence correlation spectroscopy (FCS) is frequently used to measure the self-diffusion coefficient of fluorescently labeled probes in solutions, complex media, and living cells. In a standard experiment water immersion objectives and window thickness in the range of 0.13-0.19 mm are used. We show that successful FCS measurements can be performed using samples of different refractive index placed in cells having windows of different thickness, even much thicker than nominally allowed. Different water, oil, and silicon oil immersion as well as long working distance dry objectives, equipped with the correction collar, were tested and compared. We demonstrate that the requirements for FCS experiments are less stringent than those for high resolution confocal imaging and reliable relative FCS measurements can be performed even beyond the compensation range of the objectives. All these features open new possibilities for construction of custom-made high temperature and high pressure cells for FCS.

This work investigates the interaction of carbon nitride nanosheet (CNNS), a recently developed two-dimensional nanomaterial, with DNA and its fluorescence quenching mechanism on fluorophore labeled single-stranded DNA probes. The static quenching through the photoinduced electron transfer (PET) from the excited fluorophore to the conductive band of CNNS is identified. Utilizing the affinity change of CNNS to DNA probes upon their recognition to targets and the PET-based fluorescence quenching effect, a universal sensing strategy is proposed for design of several homogeneous fluorescence detection methods with short assay time and high sensitivity. This strategy is versatile and can be combined with different amplification tools for quick fluorescence sensing of DNA and extensive DNA related analytes such as metal cations, small molecules, and proteins. As examples, two simple fluorescence detection methods for DNA and Hg(2+), one facile detection method coupled with Exo III-mediated target recycling for sensitive DNA analysis, and a ratiometric fluorescence protocol for DNA detection are proposed. This work provides an avenue for understanding the interaction between two-dimensional nanomaterials and biomolecules and designing novel sensing strategies for extending the applications of nanomaterials in bioanalysis.

In the preceding article, we introduced the theoretical framework of two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS). In this article, we report the experimental implementation of 2D FLCS. In this method, two-dimensional emission-delay correlation maps are constructed from the photon data obtained with the time-correlated single photon counting (TCSPC), and then they are converted to 2D lifetime correlation maps by the inverse Laplace transform. We develop a numerical method to realize reliable transformation, employing the maximum entropy method (MEM). We apply the developed actual 2D FLCS to two real systems, a dye mixture and a DNA hairpin. For the dye mixture, we show that 2D FLCS is experimentally feasible and that it can identify different species in an inhomogeneous sample without any prior knowledge. The application to the DNA hairpin demonstrates that 2D FLCS can disclose microsecond spontaneous dynamics of biological molecules in a visually comprehensible manner, through identifying species as unique lifetime distributions. A FRET pair is attached to the both ends of the DNA hairpin, and the different structures of the DNA hairpin are distinguished as different fluorescence lifetimes in 2D FLCS. By constructing the 2D correlation maps of the fluorescence lifetime of the FRET donor, the equilibrium dynamics between the open and the closed forms of the DNA hairpin is clearly observed as the appearance of the cross peaks between the corresponding fluorescence lifetimes. This equilibrium dynamics of the DNA hairpin is clearly separated from the acceptor-missing DNA that appears as an isolated diagonal peak in the 2D maps. The present study clearly shows that newly developed 2D FLCS can disclose spontaneous structural dynamics of biological molecules with microsecond time resolution.

The importance of fluorescent detection in many fields is well established. While advancements in instrumentation and the development of brighter fluorophore have increased sensitivity and lowered the detection limits of the method, additional gains can be made by manipulating the local electromagnetic field. Herein we take advantage of silicon nanopillars that exhibit optical resonances and field enhancement on their surfaces and demonstrate their potential in improving performance of biomolecular fluorescent assays. We use electron beam lithography and wafer scale processes to create silicon nanoscale pillars with dimensions that can be tuned to maximize fluorescence enhancement in a particular spectral region. Performance of the nanopillar based fluorescent assay was quantified using two model bioaffinity systems (biotin-streptavidin and immunoglobulin G-antibody) as well as covalent binding of fluorescently tagged bovine serum albumin (BSA). The effects of pillar geometry and number of pillars in arrays were evaluated. Color specific and pillar diameter dependent enhancement of fluorescent signals is clearly demonstrated using green and red labels (FITC, DyLight 488, Alexa 568, and Alexa 596). The ratios of the on pillar to off pillar signals normalized by the nominal increase in surface area due to nanopillars were found to be 43, 75, and 292 for the IgG-antibody assay, streptavidin-biotin system, and covalently attached BSA, respectively. Applicability of the presented approaches to the detection of small numbers of molecules was evaluated using highly diluted labeled proteins and also control experiments without biospecific analytes. Our analysis indicates that detection of fewer than 10 tagged proteins is possible.

MicroRNA (miRNA) is an important small RNA which regulates diverse gene expression at the post-transcriptional level. miRNAs are considered as important biomarkers since abnormal expression of specific miRNAs is associated with many diseases including cancer and diabetes. Therefore, it is important to develop biosensors to quantitatively detect miRNA expression levels. Here, we develop a nanosized graphene oxide (NGO) based miRNA sensor, which allows quantitative monitoring of target miRNA expression levels in living cells. The strategy is based on tight binding of NGO with peptide nucleic acid (PNA) probes, resulting in fluorescence quenching of the dye that is conjugated to the PNA, and subsequent recovery of the fluorescence upon addition of target miRNA. PNA as a probe for miRNA sensing offers many advantages including high sequence specificity, high loading capacity on the NGO surface compared to DNA and resistance against nuclease-mediated degradation. The present miRNA sensor allowed the detection of specific target miRNAs with the detection limit as low as ~1 pM and the simultaneous monitoring of three different miRNAs in a living cell.