De Novo Green Fluorescent Protein Chromophore-Based Probes for Capturing Latent Fingerprints Using a Portable System.

Rapid visualization of latent fingerprints, preferably at their point of origin, is essential for effective crime scene evaluation. Here, we present a new class of green fluorescent protein chromophore-based fluorescent dyes (LFP-Yellow and LFP-Red) that can be used for real-time visualization of LFPs within 10 s. Compared with traditional chemical reagents for LFPs, these fluorescent dyes are completely water-soluble, exhibit low cytotoxicity, and are harmless to users. Level 1-3 details of the LFPs could be clearly revealed through "off-on" fluorescence signal readout. Additionally, the fluorescent dyes were constructed based on an imidazolinone core and so do not contain pyridine groups or metal ions, which ensures that the DNA is not contaminated during extraction and identification after the LFPs are treated with the dyes. Combined with our as-developed portable system for capturing LFPs, LFP-Yellow and LFP-Red enabled the rapid capture of LFPs. Therefore, these green fluorescent protein chromophore-based probes provide an approach for the rapid identification of individuals who were present at a crime scene.

A simplified molecularly imprinted ECL sensor based on Mn2SnO4 nanocubes for sensitive detection of ribavirin.

Conventional single-signal or emerging sandwich-type double-signal electrochemiluminescence (ECL) immunosensors/aptasensors have offered accurate detection of small molecules, yet suffer from complicated setup, long processing time, and non-reusability. Here, we demonstrate a simplified molecularly imprinted ECL sensor based on Mn2SnO4 nanocubes. As an n-type semiconductor, Mn2SnO4 has numerous active sites that can capture electrons to accelerate chemical reactions, resulting in enhanced ECL activity and stability. For the first time, we verify a robust cathodic ECL emission of Mn2SnO4 luminophores in the presence of K2S2O8 coreactants. The proposed ECL sensor applies to the sensitive detection of ribavirin (RBV), endowing a wide linear range (1-2000 ng mL-1), low detection limit (0.85 ng mL-1, S/N = 3), high stability, specificity, and reproducibility, and the detection capability in real milk and chicken samples. This work highlights single semiconductor luminophore-driven molecularly imprinted ECL sensors, meeting the original aspiration of uncomplicated but high-performance sensing in food safety inspection.

Photoelectrochemical determination of diclofenac using oriented single-crystalline TiO2 nanoarray modified with molecularly imprinted polypyrrole.

A novel molecular imprint photoelectrochemical (PEC) sensor has been prepared based on oriented single-crystalline TiO2 nanoarray (TNA) material for sensitive detection of diclofenac (DCF). The TNA obtained by the one-step hydrothermal method was characterized by XRD, SEM, and TEM. Polypyrrole film was formed on the TNA by electrochemical method, and DCF was imprinted on the polymer film as the template molecule. After the removal of DCF, there appeared lots of specific recognition sites that matched template molecules. The experimental results demonstrated that the constructed PEC sensor has good sensitivity and selectivity for the detection of DCF, which can be attributed to the high photoelectric conversion efficiency of TNA and the high selectivity of molecular imprinting technology. The fabricated PEC sensor showed a wide detection range (0.05-1000 µM) and a low limit of detection (0.0034 µM) for DCF, as well as good repeatability and stability. The proposed PEC sensor provided an effective strategy in the monitoring of environmental pollutants.

A new long-wavelength fluorescent probe for tracking peroxynitrite in live cells and inflammatory sites of zebrafish.

Peroxynitrite (OONO-), as a reactive oxygen species (ROS), would be mostly profoundly implicated in diseases such as inflammation in organisms. However, bioimaging of ONOO- still faces difficulties owing to the shortage of bioimaging and real-time dynamic tracking distribution of ROS in inflammation. To address this challenge, we designed and synthesized a long-wavelength fluorescent probe based on tricyanofuran (ACDM-BE), which exhibits a fast response (response time is 40 s), high selectivity and great sensitivity (LOD is approximately 21 nM) towards ONOO-. ACDM-BE was shown to be capable of detecting ONOO- in living cells and monitor the changes in ONOO- levels under the stimulus of various concentrations of SIN-1 (from 100 to 700 µM), which was successfully tracked by the fluorescence changes in live cells. It is worth noting that ACDM-BE further demonstrated its ability to track the dynamic changes of the level of ONOO- in the inflammatory sites of larval zebrafish. Thus, ACDM-BE could be employed as an efficient tool for exploiting the role of ONOO- in inflammation in living biosystems.

Ultrasensitive Electrochemical Biosensor of Bacterial 16S rRNA Gene Based on polyA DNA Probes.

Traditional microbiology analysis is usually hindered by the long time-cost and lack of portability in many urgent situations. In this work, we developed a novel electrochemical DNA biosensor (E-biosensor) for sensitive analysis of the 16S rRNA gene of five bacteria, using a consecutive adenine (polyA) probe. The polyA probe consists of a polyA tail and a recognition part. The polyA tail can combine onto the gold surface with improved controllability of the surface density, by conveniently changing the length of polyA. The recognition part of the capture probe together with two biotin-labeled reporter probes hybridize with the target DNA and form a stable DNA-tetramer sandwich structure, and then avidin-HRP enzyme was added to produce a redox current signal for the following electrochemical detection. Finally, we realized sensitive quantification of artificial target DNA with a limit of detection (LOD) of 10 fM, and excellent selectivity and reusability were also demonstrated. Importantly, the detection capability was equally good when facing bacterial genomic DNA, due to the base-stacking force of our multireporter-probe system, which can help to break the second structure and stabilize the probe-target complexes. Our biosensor was constructed on a 16-channel electrode chip without any polymerase chain reaction (PCR) process needed, which took a significant step toward a portable bacteria biosensor.

A nanocomposite prepared from hemin and reduced graphene oxide foam for voltammetric sensing of hydrogen peroxide.

A foam consisting of reduced graphene oxide was synthesized by a one-pot hydrothermal method. The foam was used to prepare a nanocomposite with hemin which is formed via π-interactions. The nanocomposite was incorporated via a Nafion film and then placed on a glassy carbon electrode (GCE). The modified GCE displays outstanding catalytic activity towards H2O2. It is assumed that this is due to (a) the redox-active center [Fe(III/II)] of hemin, and (b) the crosslinked macroporous structure of the foam. Both improve the electron transfer rate and electrochemical signals. Under the optimum experimental conditions and a working voltage of typically -0.41 mV (vs. SCE), the sensor has a 2.8 nM H2O2 detection limit, and the analytically useful range extends from 5 nM to 5 mM with a sensitivity of 50.5 µA µM-1 cm-2. The modified GCE has high sensitivity and fast response. It was utilized to quantify H2O2 in spiked environmental water samples. Graphical abstractSchematic representation of the electrochemical sensor based on a nanocomposite prepared from hemin and reduced graphene oxide foam, which can be applied to the determination of hydrogen peroxide in serum.

A FRET-based fluorescent probe for hydrogen peroxide based on the use of carbon quantum dots conjugated to gold nanoclusters.

In the Fenton reaction, ferrous ion acts as a catalyst and reacts with hydrogen peroxide (H2O2) to produce hydroxy radicals (·OH) and hydroperoxy radicals (·OOH). Both have much stronger oxidization ability than H2O2. A fluorescent probe for H2O2 is described here that was obtained by covalent conjugation of carbon quantum dots to gold nanoclusters (AuNCs). The conjugate, under 360 nm photoexcitation, displays dual (blue and red) emission, with peaks located at 450 and 640 nm. When introducing ·OH radicals via the Fenton reaction, the fluorescence intensities of both the CQDs and the AuNCs are decreased. The ratio of the fluorescence at the two peaks is related to the concentration of H2O2 in the 1.25 nM to 10 µM concentration range, and the detection limit is 0.16 nM. The probe was applied to the determination of H2O2 in milk and toothpaste and to cell imaging. Graphical abstract Schematic diagram of the FRET-based fluorescent probe and enhanced performance of hydrogen peroxide detection via Fenton reaction. The fluorescence intensity of CQD-AuNCs nanoaster was decreased as introducing H2O2 to the probe, and can be applied to the determination of milk and toothpaste and cells imaging.

Photoelectrochemical sensing of dopamine using gold-TiO2 nanocomposites and visible-light illumination.

A photoelectrochemical (PEC) method was developed for the determination of dopamine. It is making use of a composite prepared from gold nanoparticles and TiO2 (type P25) and placed on a fluorine-doped tin oxide (FTO) electrode. The composites are used for photoelectrical detection with improved electron transfer efficiency for photoproduction and with improved photoelectrical conversion efficiency. This is due to the excellent electrical conductivity and surface plasmon resonance absorption by gold nanoparticles, and also by the photocatalytic effect of TiO2. Dopamine binds easily to the surface of the composites and acts as an electron donor. This electrode gives a strongly enhanced photocurrent which increases linearly in the 0.1 to 100 µM dopamine concentration range and has a 23 nM detection limit (at S/N = 3). The electrode was operated over 15 cycles of light-on and light-off states every 20 s under visible-light illumination, and the sensor indicates good stability. In addition, it is selective over several possible interferents including uric acid, L-cysteine, glutathione, ascorbic acid and glucose. Graphical abstract A new gold/P25 composite-based photoelectrochemical sensing scheme for dopamine is described. Under visible light irradiation, the photocurrent response is increased with the increasing concentration of dopamine (DA).

Correction to: Photoelectrochemical sensing of dopamine using gold-TiO2 nanocomposites and visible-light illumination.

The published version of this article, unfortunately, contains error. The author found out that Chinese characters are shown in Scheme 1a.

3D carbon nanosphere and gold nanoparticle-based voltammetric cytosensor for cell line A549 and for early diagnosis of non-small cell lung cancer cells.

An electrochemical cytosensor for the detection of the non-small-cell lung cancer cell line A549 (NSCLC) had been developed. A microwave-hydrothermal method was employed to prepare monodisperse colloidal carbon nanospheres (CNSs). Gold nanoparticles (AuNPs) were placed on the surface of the colloidal CNSs by self-assembly to obtain 3D-structured microspheres of the type CNS@AuNP. The results of an MTT assay show the microspheres to possess good biocompatibility. The CNS@AuNP nanocomposite was then placed, in a chitosan film, on a glassy carbon electrode (GCE). The voltammetric signals and detection sensitivity are significantly enhanced owing to the synergistic effect of CNSs and AuNPs. A cytosensor was then obtained by immobilization of antibody against the carcinoembryonic antigen (which is a biomarker for NSCLC) on the GCE via crosslinking with glutaraldehyde. Hexacyanoferrate is used as an electrochemical probe, and the typical working voltage is 0.2 V (vs. SCE). If exposed to A549 cells, the differential pulse voltammetric signal decreases in the 4.2 × 10-1 to 4.2 × 10-6 cells mL-1 concentration range, and the detection limit is 14 cells mL-1 (at S/N = 3). Graphical abstract Schematic presentation of design strategy and fabrication process of the electrochemical cytosensor for A549 cells. (CNS: carbon nanospheres; GA: glutaraldehyde; PEI: polyethyleneimine; AuNPs: gold nanoparticles; BSA: Bovine serum albumin).

Hyaluronic acid-grafted three-dimensional MWCNT array as biosensing interface for chronocoulometric detection and fluorometric imaging of CD44-overexpressing cancer cells.

A sandwich-type electrochemical cytosensor is described for quantitative determination of CD44-overexpressing HeLa cells. Hyaluronic acid (HA) acts as a targeting molecule that was in-situ incorporated into the sensor based on the use of an indium tin oxide (ITO) electrode modified with multi-walled carbon nanotubes (MWCNTs). The 3D-MWCNT structure is shown to strongly improve the electronic properties and surface chemical reactivities. The HA-modified sensor exhibits a highly sensitive response to HeLa cells. A sandwiched hybridization protocol was then established using BIO [an N-butyl-4-(6'-aminohexyl)amino-1,8-naphthalimide probe modified with HA] as the tracing labels of the fluorescent probes for targeting CD44-positive tumor cells. The signal amplification was thereby maximized and measured by chronocoulometry. The binding of CD44-positive HeLa cells to the HA modified sensing layer causes a decrease in chronocoulometric response. The signal decreases linearly in the 2.1 × 102 to 2.1 × 107 HeLa cells·mL-1 concentration range with a detection limit of 70 cells·mL-1. Such a sandwich-type assay may be tailored as a sensitive candidate for detecting low levels of tumor cells. Graphical abstract Schematic of a sandwich cytosensor based on hyaluronic acid-grafted 3D-MWCNT as biosensing interface and BIO as fluorescent probe. This biosensor possessed excellent electrochemical activity, high sensitivity and selectivity, providing a dynamical tracking and detecting platform for CD44-positive tumor cells.

An ultrasensitive multi-walled carbon nanotube-platinum-luminol nanocomposite-based electrochemiluminescence immunosensor.

An ultrasensitive electrochemiluminescence (ECL) immunosensor for carbohydrate antigen 19-9 (CA19-9) detection using multi-walled carbon nanotube-platinum-luminol nanocomposites (MWCNT-Pt-luminol) as nanointerface and signal tags was designed. First, the MWCNT-Pt-luminol nanocomposite was decorated on the surface of a glassy carbon electrode (GCE) through the film-forming properties of chitosan (Chi). Then, the CA19-9 antibody (anti-CA19-9) was attached to the modified electrode surface via crosslinking with glutaraldehyde (GA). When CA19-9 was captured by its antibody immobilized on the immune platform via immunoreaction, the ECL signal intensity decreased. Under optimal conditions, the proposed ECL immunosensor showed excellent performance for CA19-9 ranging from 0.0001 U mL-1 to 10.0 U mL-1, with a detection limit of 0.000046 U mL-1 (S/N = 3) and a correlation coefficient of R = 0.9980. This is attributed to the fact that the MWCNTs-Pt nanomaterial has excellent conductivity and it could facilitate the decomposition of H2O2 to generate various reactive oxygen species (ROSs); thus, the ECL signals of luminol were effectively amplified and the sensitivity of the sensor was greatly increased. The prepared ECL immunosensor displayed simple, fast analysis, excellent stability, good reproducibility, and high specificity. Moreover, the developed ECL immunosensor provided satisfactory results in the determination of CA19-9 in real human serum samples.

Dual-Modal Colorimetric/Fluorescence Molecular Probe for Ratiometric Sensing of pH and Its Application.

As traditional pH meters cannot work well for minute regions (such as subcellular organelles) and in harsh media, molecular pH-sensitive devices for monitoring pH changes in diverse local heterogeneous environments are urgently needed. Here, we report a new dual-modal colorimetric/fluorescence merocyanine-based molecular probe (CPH) for ratiometric sensing of pH. Compared with previously reported pH probes, CPH bearing the benzyl group at the nitrogen position of the indolium group and the phenol, which is used as the acceptor for proton, could respond to pH changes immediately through both the ratiometric fluorescence signal readout and naked-eye colorimetric observation. The sensing process was highly stable and reversible. Most importantly, the suitable pKa value (6.44) allows CPH to presumably accumulate in lysosomes and become a lysosome-target fluorescent probe. By using CPH, the intralysosomal pH fluctuation stimulated by antimalaria drug chloroquine was successfully tracked in live cells through the ratiometric fluorescence images. Additionally, CPH could be immobilized on test papers, which exhibited a rapid and reversible colorimetric response to acid/base vapor through the naked-eye colorimetric analysis. This proof-of-concept study presents the potential application of CPH as a molecular tool for monitoring intralysosomal pH fluctuation in live cells, as well as paves the way for developing the economic, reusable, and fast-response optical pH meters for colorimetric sensing acid/base vapor with direct naked-eye observation.

A Functional CT Contrast Agent for In Vivo Imaging of Tumor Hypoxia.

Hypoxia, which has been well established as a key feature of the tumor microenvironment, significantly influences tumor behavior and treatment response. Therefore, imaging for tumor hypoxia in vivo is warranted. Although some imaging modalities for detecting tumor hypoxia have been developed, such as magnetic resonance imaging, positron emission tomography, and optical imaging, these technologies still have their own specific limitations. As computed tomography (CT) is one of the most useful imaging tools in terms of availability, efficiency, and convenience, the feasibility of using a hypoxia-sensitive nanoprobe (Au@BSA-NHA) for CT imaging of tumor hypoxia is investigated, with emphasis on identifying different levels of hypoxia in two xenografts. The nanoprobe is composed of Au nanoparticles and nitroimidazole moiety which can be electively reduced by nitroreductase under hypoxic condition. In vitro, Au@BSA-NHA attain the higher cellular uptake under hypoxic condition. Attractively, after in vivo administration, Au@BSA-NHA can not only monitor the tumor hypoxic environment with CT enhancement but also detect the hypoxic status by the degree of enhancement in two xenograft tumors with different hypoxic levels. The results demonstrate that Au@BSA-NHA may potentially be used as a sensitive CT imaging agent for detecting tumor hypoxia.

Electrochemical detection of PCR amplicons of Escherichia coli genome based on DNA nanostructural probes and polyHRP enzyme.

Fast, portable and sensitive analysis of E. coli is becoming an important challenge in many critical fields (e.g., food safety, environmental monitoring and clinical diagnosis). Thus, electrochemical biosensing of PCR amplicons from the bacterial genome has attracted reasonable research attention. In this work, we utilized a 3D DNA tetrahedral probe to establish a "sandwich-type" electrochemical DNA biosensor for sensitive and specific analysis of a 250 bp unpurified PCR amplicon from the uidA gene of the E. coli genome. Asymmetric PCR was used to produce single-stranded PCR products. Streptavidin-polyHRP80 was employed to improve the signal gain during electrochemical detection. We optimized important experimental conditions for DNA sensing, including the streptavidin-polyHRP, the signal probe and the ion strength. Finally, we achieved a remarkable sensitivity of 10 fM synthetic DNA target, and successfully performed the analysis of PCR amplicons from as low as 0.2 pg µL(-1) of E. coli genome. Compared with traditional single stranded DNA (ssDNA) probe based detection, our present work demonstrated 3 orders of magnitude improvement in sensitivity. In addition, our electrochemical DNA biosensor was 4 orders of magnitude more sensitive than normal electrophoretic analysis of PCR products. Our work made important progress in DNA nanostructured probe-based biosensors toward application in real applications.

Multiwalled carbon nanotubes/gold nanocomposites-based electrochemiluminescent sensor for sensitive determination of bisphenol A.

An electrochemiluminescence (ECL) sensor for bisphenol A was proposed by using L-cysteine-functionalized multiwalled carbon nanotubes/gold nanocomposites-modified glassy carbon electrode (MWCNTs-Au/GCE) based on ECL of peroxydisulfate solution. The ECL behaviors of peroxydisulfate solution had been investigated at the chitosan/MWCNTs-Au/GCE, and bisphenol A was found to have quenching effects on the ECL of peroxydisulfate solution. Both Au nanoparticles (AuNPs) and multiwalled CNTs could promote the electron transfer and synergetically amplify the ECL signal of peroxydisulfate solution. Under the optimized conditions, the ECL signal intensity was linear with the concentration of bisphenol A in the concentration range between 0.25 and 100 µM (R = 0.9931) with a detection limit (S/N = 3) of 0.083 µM. The constructed ECL sensor has the advantages of simplicity, sensitivity, good selectivity, and reproducibility, exhibiting a great potential application in the determination of bisphenol A.

Selective and ratiometric fluorescent trapping and quantification of protein vicinal dithiols and in situ dynamic tracing in living cells.

Protein vicinal dithiols play fundamental roles in intracellular redox homeostasis due to their involvement in protein synthesis and function through the reversible vicinal dithiol oxidation to disulfide. To provide quantitative information about the global distribution and dynamic changes of protein vicinal dithiols in living cells, we have designed and synthesized a ratiometric fluorescent probe (VTAF) for trapping of vicinal dithiol-containing proteins (VDPs) in living cells. VTAF exhibits a ratiometric fluorescence signal upon single excitation, which enables self-calibration of the fluorescence signal and quantification of endogenous vicinal dithiols of VDPs. Its potential for in situ dynamic tracing of changes of protein vicinal dithiols under different cellular redox conditions was exemplified. VTAF facilitated the direct observation of subcellular distribution of endogenous VDPs via ratiometric fluorescence imaging and colocalization assay. And the results suggested that there are abundant VDPs in mitochondria. Moreover, some redox-sensitive VDPs are also present on cell surface which can respond to redox stimulus. This ratiometric fluorescence technique presents an important extension to previous fluorescence intensity-based probes for trapping and quantifying protein vicinal dithiols in living cells, as well as its visible dynamic tracing of VDPs.

Multi-channel colorimetric and fluorescent probes for differentiating between cysteine and glutathione/homocysteine.

Three fluorescent probes TP1­3 for thiols were rationally designed and synthesized to distinguish cysteine (Cys) from glutathione (GSH)/homocysteine (Hcy). TP1­3 are almost non-fluorescent and colorless 4-nitro-1,8-naphthalimide derivatives. Upon the substitution of nitro by Cys, TP1­3 were transformed into weakly fluorescent green-emitting 4-amino analogs via highly fluorescent blue-emitting thioether intermediates. The three-channel signaling capability allows discrimination between Cys and GSH/Hcy. The fluorescence intensity at 498 nm was linearly proportional to GSH concentration in the range of 0-20 µM, and the detection limit was 5 × 10(-8) mol L(-1). A good linear relationship between A446/A350 and Cys concentration was found in the range of 0-70 µM, and the detection limit was 2 × 10(-7) mol L(-1). Moreover, TP3 was used for living cell imaging as well as for detecting mercapto-containing proteins.

A colloidal gold nanoparticle-based immunochromatographic test strip for rapid and convenient detection of Staphylococcus aureus.

An immunochromatographic test strip using gold nanoparticles-staphylococcus aureus monoclonal antibody conjugates was developed for the rapid and convenient detection of staphylococcus aureus based on a double-antibody sandwich format. The detection limit and the detection rate of this test strip is 10(3) CFU /mL and 98.7%, respectively. It could be used for the rapid detection of staphylococcus aureus in food and the results can be visually identified by the naked eye within 10 min. Compared with conventional bacterial detection methods, this developed immunochromatographic assay based test strip has several advantages including simple, fast, low-cost, favorable sensitivity and specificity, exhibiting a great potential for application in food safety control systems and clinical diagnosis.

N-doped TiO2/Ti3C2-driven self-photocatalytic molecularly imprinted ECL sensor for sensitive and steady detection of dexamethasone.

The conventional luminol-based electrochemiluminescence (ECL) biosensor suffers from hampered signal stability due to the self-decomposition of the H2O2 co-reactant. Here, we propose an N-doped TiO2/Ti3C2 heterojunction driven self-photocatalytic platform for ECL signal amplification and then combine it with molecular imprinting technology for sensitive and steady detection of dexamethasone (DXM). Unlike traditional cases involving specific catalysts or external electron injection, the initial luminescence of luminol in this new system is utilized as the excitation light of N-doped TiO2/Ti3C2 photocatalyst to promote the conversation of dissolved oxygen to H2O2, supplying more co-reactants to improve ECL of luminol in turn. Thanks to the heterojunction and self-photocatalytic cyclic amplification, this molecularly imprinted ECL sensor exhibits a wide linear range (1.0 × 10-6-1.0 × 101 µg mL-1) and a low detection limit, as well as excellent anti-interference capability, sensitivity, and stability. This work contributes to more reliable and steady detection of DXM and brings new insights into developing exogenous co-reactant-free self-enhancement ECL models for biosensor applications.