Single Molecule-Level Detection via Liposome-Based Signal Amplification Mass Spectrometry Counting Assay.

Because of the low atomization and/or ionization efficiencies of many biological macromolecules, the application of mass spectrometry to the direct quantitative detection of low-abundance proteins and nucleic acids remains a significant challenge. Herein, we report mass spectrum tags (MS-tags) based upon gold nanoparticle (AuNP)-templated phosphatidylcholine phospholipid (DSPC) liposomes, which exhibit high and reliable signals via electrospray ionization (ESI). Using these MS-tags, we constructed a liposome signal amplification-based mass spectrometric (LSAMS) "digital" counting assay to enable ultrasensitive detection of target nucleic acids. The LSAMS system consists of liposomes modified with a gold nanoparticle core and surface-anchored photocleavable DNA. In the presence of target nucleic acids, the modified liposome and a magnetic bead simultaneously hybridize with the target nucleic acid. After magnetic separation and photolysis, the MS-tag is released and can be analyzed by ESI-MS. At very low target concentrations, one liposome particle corresponds to one target molecule; thus, the concentration of the target can be estimated by counting the number of liposomes. With this assay, hepatitis C (HCV) virus RNA was successfully analyzed in clinical samples.

Multivalent Self-Assembled DNA Polymer for Tumor-Targeted Delivery and Live Cell Imaging of Telomerase Activity.

The efficient detection and in situ monitoring of telomerase activity is of great importance for cancer diagnosis and biomedical research. Here we report for the first time that the development of a novel multivalent self-assembled DNA polymer, constructed through telomerase primer sequence (ITS) triggered hybridization chain assembly using two functional hairpin probes (tumor-trageting aptamer modified H1 and signal probe modified H2), for sensitive detection and imaging of telomerase activity in living cells. After internalizing into the tumor cells by multivalent aptamer targeting, the ITS on DNA polymers can be elongated by intracellular telomerase to generate telomere repeat sequences that are complementary with the signal probe, which can proceed along the DNA polymers, and gradually light up the whole DNA polymers, leading to an enhanced fluorescence signal directly correlated with the activity of telomerase. Our results demonstrated that the developed DNA polymer show excellent performance for specifically detecting telomerase activity in cancer cells, dynamically monitoring the activity change of telomerase in response to telomerase-based drugs, and efficiently distinguishing cancer cells from normal cells. The proposed strategy may afford a valuable tool for the monitoring of telomerase activity in living cells and have great implications for biological and diagnostic applications.

Core-Shell-Shell Multifunctional Nanoplatform for Intracellular Tumor-Related mRNAs Imaging and Near-Infrared Light Triggered Photodynamic-Photothermal Synergistic Therapy.

A multifunctional nanoplatform, which generally integrates biosensing, imaging diagnosis, and therapeutic functions into a single nanoconstruct, has great important significance for biomedicine and nanoscience. Here, we developed a core-shell-shell multifunctional polydopamine (PDA) modified upconversion nanoplatform for intracellular tumor-related mRNAs detection and near-infrared (NIR) light triggered photodynamic and photothermal synergistic therapy (PDT-PTT). The nanoplatform was constructed by loading a silica shell on the hydrophobic upconversion nanoparticles (UCNPs) with hydrophilic photosensitizer methylene blue (MB) entrapped in it, and then modifying PDA shells through an in situ self-polymerization process, thus yielding a core-shell-shell nanoconstruct UCNP@SiO2-MB@PDA. By taking advantages of preferential binding properties of PDA for single-stranded DNA over double-stranded DNA and the excellent quenching property of PDA, a UCNP@SiO2-MB@PDA-hairpin DNA (hpDNA) nanoprobe was developed through adsorption of fluorescently labeled hpDNA on PDA shells for sensing intracellular tumor-related mRNAs and discriminating cancer cells from normal cells. In addition, the fluorescence resonance energy transfer from the upconversion fluorescence (UCF) emission at 655 nm of the UCNPs to the photosensitizer MB molecules could be employed for PDT. Moreover, due to the strong NIR absorption and high photothermal conversion efficiency of PDA, the UCF emission at 800 nm of the UCNPs could be used for PTT. We demonstrated that the UCNP@SiO2-MB@PDA irradiated with NIR light had considerable PDT-PTT effect. These results revealed that the developed multifunctional nanoplatform provided promising applications in future oncotherapy by integrating cancer diagnosis and synergistic therapy.

Melanin-Like Nanoquencher on Graphitic Carbon Nitride Nanosheets for Tyrosinase Activity and Inhibitor Assay.

Graphitic C3N4 (g-C3N4) nanosheets are a type of emerging graphene-like carbon-based nanomaterials with high fluorescence and large specific surface areas that hold great potential for biosensor applications. However, current g-C3N4 based biosensors have prevailingly been limited to coordination with metal ions, and it is of great significance to develop new designs for g-C3N4 nanosheets based biosensors toward biomarkers of general interest. We report the development of a novel g-C3N4 nanosheet-based nanosensor strategy for highly sensitive, single-step and label-free detection of tyrosinase (TYR) activity and its inhibitor. This strategy relies on the catalytic oxidation of tyrosine by TYR into melanin-like polymers, which form a nanoassembly on the g-C3N4 nanosheets and quench their fluorescence. This strategy was demonstrated to provide excellent selectivity and superior sensitivity and to enable rapid screening for TYR inhibitors. Therefore, the developed approach might create a useful platform for diagnostics and drugs screening for TYR-based diseases including melanoma cancer.

Electrochemical immunosensor based on Pd-Au nanoparticles supported on functionalized PDDA-MWCNT nanocomposites for aflatoxin B1 detection.

This paper reports a label-free electrochemical immunosensor for the determination of aflatoxin B1 (AFB1), which is based on a gold electrode modified by a biocompatible film of carbon nanotubes/poly(diallyldimethylammoniumchloride)/Pd-Au nanoparticles (CNTs/PDDA/Pd-Au). The nanocomposite was characterized by transmission electron microscopy and the electrochemical behavior of modified electrodes was investigated by cyclic voltammetry. The CNTs/PDDA/Pd-Au nanocomposites film showed good electron transfer ability, which ensured high sensitivity to detect AFB1 in a range from 0.05 to 25 ng mL(-1) with a detection limit of 0.03 ng mL(-1) obtained at 3σ (where σ is the standard deviation of the blank solution, n = 10). The proposed immunosensor provides a simple tool for AFB1 detection. This strategy can be extended to any other antigen detection by using the corresponding antibodies.

Label-free fluorescence detection of microRNA based on target induced adenosine2-coralyne-adenosine2 formation.

This study develops a simple and label-free biosensor for sensitive and selective detection of microRNA (miRNA) based on the formation of the adenosine2-coralyne-adenosine2 complex mediated by miRNA-specific polyadenosine extension.

Phospholipid-modified upconversion nanoprobe for ratiometric fluorescence detection and imaging of phospholipase D in cell lysate and in living cells.

Phospholipase D (PLD) is a critical component of intracellular signal transduction and has been implicated in many important biological processes. It has been observed that there are abnormalities in PLD expression in many human cancers, and PLD is thus recognized as a potential diagnostic biomarker as well as a target for drug discovery. We report for the first time a phospholipid-modified nanoprobe for ratiometric upconversion fluorescence (UCF) sensing and bioimaging of PLD activity. The nanoprobe can be synthesized by a facile one-step self-assembly of a phospholipid monolayer composed of poly(ethylene glycol) (PEG)ylated phospholipid and rhodamine B-labeled phospholipid on the surface of upconversion nanoparticles (UCNPs) NaYF4: 20%Yb, 2%Er. The fluorescence resonance energy transfer (FRET) process from the UCF emission at 540 nm of the UCNPs to the absorbance of the rhodamine B occurs in the nanoprobe. The PLD-mediated hydrolysis of the phosphodiester bond makes rhodamine B apart from the UCNP surface, leading to the inhibition of FRET. Using the unaffected UCF emission at 655 nm as an internal standard, the nanoprobe can be used for ratiometric UCF detection of PLD activity with high sensitivity and selectivity. The PLD activity in cell lysates is also determined by the nanoprobe, confirming that PLD activity in a breast cancer cell is at least 7-fold higher than in normal cell. Moreover, the nanoprobe has been successfully applied to monitoring PLD activity in living cells by UCF bioimaging. The results reveal that the nanoprobe provides a simple, sensitive, and robust platform for point-of-care diagnostics and drug screening in biomedical applications.

Hydroxyapatite nanoarray-based cyanide biosensor.

Here we report a simple, biomolecular-friendly protocol for the fabrication of a hydroxyapatite nanowires array (HANWA) biosensor of spatial positioning, large surface area, and abundant adsorbing sites and its application to cyanide sensing. The fabrication of HANWA is performed by template-assisted electrodeposition. The well-aligned hydroxyapatite nanoarray is composed of vertical nanowires with a diameter of approximately 200 nm and an average length of 1 microm. The electrochemical biosensor for the determination of cyanide through its inhibitory effect on horseradish peroxidase (HRP) encapsulated by chitosan (CHIT) on the platform of HANWA is demonstrated. The current organic-inorganic hybrid nanostructure provides excellent enzyme-substrate contact with enzyme activity well maintained. The densely distributed HANWA with large surface area and abundant adsorbing sites can provide a favorable electrochemical interface for the construction of electrochemical biosensor. A sensitive detection limit of 0.6 ng ml(-1) was obtained for cyanide. The proposed CHIT-HRP/HANWA biosensor has the advantages of spatial resolution, high sensitivity, rapid regeneration, and fast response associated with individual nanowires. It broadens the possible applications of chemosensors and biosensors, and it offers an alternative method for toxic substance determination. The new device holds great promise for environmental and food industrial monitoring of toxins.

DNA encapsulating liposome based rolling circle amplification immunoassay as a versatile platform for ultrasensitive detection of protein.

A novel rolling circle amplification (RCA) immunoassay based on DNA-encapsulating liposomes, liposome-RCA immunoassay, was developed for ultrasensitive protein detection. This technique utilized antibody-modified liposomes with DNA prime probes encapsulated as the detection reagent in the sandwiched immunoassays. The DNA prime probes were released from liposomes and then initiated a linear RCA reaction, generating a long tandem repeated sequences that could be selectively and sensitively detected by a microbead-based fluorescence assay. The developed technique offered very high sensitivity due to primary amplification via releasing numerous DNA primers from a liposome followed by a secondary RCA amplification. A biobarcode design was incorporated in the technique, which allowed the strategy to be directly implemented for multiplex assay of multiple proteins. Also, the technique allowed easy preparation of the DNA-carrying antibody reagent and the implementation with simple instrumentation. The technique was demonstrated for the determination of prostate-specific antigen (PSA), a highly selective biomarker associated with prostate cancer. The results revealed that the technique exhibited a dynamic response to PSA over a 6-decade concentration range from 0.1 fg mL(-1) to 0.1 ng mL(-1) with a limit of detection as low as 0.08 fg mL(-1) and a high dose-response sensitivity. The liposome-RCA immunoassay holds great promise as a versatile, sensitive, and robust platform to combine the nucleic acid amplification with immunoassay for ultrasensitive protein detection.

Surface-enhanced Raman spectroscopic detection of a bacteria biomarker using gold nanoparticle immobilized substrates.

The development of ultrasensitive and rapid methods for the detection of dipicolinic acid (DPA), a biomarker for bacterial spores including Bacillus anthracis, is increasingly important. This paper reports the results of an investigation of surface enhanced Raman spectroscopy (SERS) based ultrasensitive detection of DPA using a gold nanoparticle/polyvinylpyrrolidone/gold substrate (AuNPs/PVP/Au). The strong SERS effect of this substrate exploits the particle-particle and particle-substrate plasmonic coupling, which is optimized by manipulating the diameter of the nanoparticles (50-70 nm). The correlation between the SERS intensity of the diagnostic band and the DPA concentration (0.1 ppb to 100 ppm) was shown to exhibit two linear regions, i.e., the low- (<0.01 ppm) and high-concentration (>1 ppm) regions, with an intermediate region in between. The presence of a linear relationship in the low-concentration region was observed for the first time in SERS detection of DPA. A detection limit of 0.1 ppb was obtained from the substrates with 60 nm sized Au NPs, which is, to our knowledge, the lowest detection limit reported for DPA using this type of SERS substrate. This finding was also supported by the estimated enhancement factor (approximately 10(6)) and a large adsorption equilibrium constant for the low-concentration region (1.7 x 10(7) M(-1)). The adsorption characteristics of DPA on the SERS substrates were analyzed in terms of monolayer and multilayer adsorption isotherms to gain insights into the correlation between the SERS intensity and the DPA concentration. The observed transition from the low- to high-concentration linear regions was found to correspond to the transition from a monolayer to multilayer adsorption isotherm, which was in agreement with the estimated minimum DPA concentration for a monolayer coverage (approximately 0.01 ppm).

Generalized ratiometric fluorescence nanosensors based on carbon dots and an advanced chemometric model.

Probe encapsulated by biologically localized embedding (PEBBLE) has emerged as a new type of sensing technique for complex systems. Generalized ratiometric PEBBLE nanosensors prepared by encapsulating an intensity-based probe and an inert reference dye inside the pores of stable matrix possess advantages of easy synthesis, immunity to interference, lower toxicity, and robustness to variations in probe loading. However, the selection of appropriate reference dyes used in generalized ratiometric PEBBLE nanosensors is a rather difficult task since they should satisfy some stringent requirements. In this contribution, the feasibility of using carbon dots (C-dots) as generic inert references in synthesizing PEBBLE nanosensors was first investigated in detail. And a dual-wavelength monitoring strategy and the quantitative fluorescence model for generalized ratiometric probes (QFMGRP) were adopted to solve the problems brought by the use of carbon dots as inert references. C-dots doped PEBBLE nanosensors (C-PEBBLE nanosensors) for the quantification of NO2- and free Ca2+ were synthesized by encapsulating C-dots and intensity based fluorescence probes (i.e., acriflavine for NO2-, and Rhod-2 for Ca2+, respectively) inside the pores of stable matrix. Experimental results showed that the combination of C-PEBBLEs, the QFMGRP model and the dual-wavelength monitoring strategy achieved accurate quantification of NO2- and the free Ca2+ in real-world samples. Their quantitative results were in good consistence with those determined by HPLC and atomic absorption spectrophotometer, respectively. The strategies proposed in this contribution have generic applicability in the synthesis of PEBBLE nanosensors and their quantitative applications.

Sensitive fluorescence sensing of T4 polynucleotide kinase activity and inhibition based on DNA/polydopamine nanospheres platform.

5'-Polynucleotide kinase (PNK) is a crucial enzyme that catalyzes the phosphorylation of nucleic acid with 5'-OH termini and this phosphorylation reaction has been involved in many important cellular activities. The evaluation of PNK activity has received an increasing attention due to the significance of PNK. Here, the polydopamine nanospheres (PDANS) could adsorb single-stranded DNA (ssDNA) through π-π stacking or hydrogen bonding between nucleobases and aromatic groups of PDANS, while the interaction between double-stranded DNA (dsDNA) with PDANS was weakened due to the changed conformation. Hence, a novel DNA/PDANS platform was constructed for the sensitive and selective determination of T4 PNK activity based on the preferential binding properties of PDANS for ssDNA over dsDNA and the excellent fluorescence quenching property of PDANS. The dye-labeled dsDNA was phosphorylated by T4 PNK and then digested by λ exonuclease, yielding dye-labeled ssDNA, which would be adsorbed on the surface of the PDANS and the fluorescence was greatly quenched by PDANS. Because of the preferential binding properties of PDANS for ssDNA over dsDNA and the high quenching property of PDANS, the developed DNA/PDANS platform exhibited good analytical performance for T4 PNK sensing in complex biological matrix and applied to screening inhibitors. The proposed DNA/PDANS based platform is promising in developing high-throughput assays for drug screening and clinical diagnostics.

A novel piezoelectric immunoagglutination assay technique with antibody-modified liposome.

A simple rapid piezoelectric immunoagglutination assay (PEIA) technique with antibody-modified liposome has been developed for direct quantitative detection of human immunoglobulin G (hIgG). This technique is based on specific agglutination of antibody-coated liposome particles in the presence of the corresponding antigen, which can be monitored by the frequency shift of a piezoelectric device. Compared with conventional piezoelectric assays, this liposome-based PEIA does not require the immobilization of antigen or antibody on the quartz crystal surface, making the developed technique especially useful for rapid and renewable immunochemical determination. To alleviate non-specific adsorption of serum proteins, modification of the quartz crystal surface by different protocols and the composition of the assay medium have been investigated. The results indicate that the background interference can be substantially minimized through modifying the quartz crystal surface with a bovine serum albumin (BSA) layer and introducing an appropriate amount of BSA in the assay medium. The effects of the liposome composition, the liposome concentration and the concentration of poly(ethylene glycol) (PEG) in the assay medium, have also been investigated. The frequency responses of the liposome-based PEIA are linearly correlated to hIgG concentration in the range of 0.05-6 microg mL(-1) with a detection limit of 50 ng mL(-1).

A direct electrochemical biosensing platform constructed by incorporating carbon nanotubes and gold nanoparticles onto redox poly(thionine) film.

A direct electrochemical biosensing platform has been fabricated by covalent incorporation of carbon nanotubes (CNT) and gold nanoparticles (GNP) onto the poly(thionine) (PTH) film deposited by electropolymerization. With the synergic effects of the composite nanomaterials together with the excellent mediating redox polymer, the proposed platform could allow for faster electron transfer and higher enzyme immobilization efficiency than the platforms designed by using CNT or GNP alone. Comparison studies indicated that the as-developed H(2)O(2) sensor could show greatly improved performances of amperometric responses.

An optode sensor for Cu2+ with high selectivity based on porphyrin derivative appended with bipyridine.

A porphyrin derivative (fluorophore) appended with bipyridine (ionophore) has been applied for preparation of a Cu2+-sensitive optical chemical sensor, which is based on fluorescence quenching of porphyrin derivative entrapped in a poly(vinyl chloride) membrane by the energy transfer process. The sensor exhibits a linear response toward Cu2+ in the concentration range 2.0 x 10(-8) - 1.0 x 10(-5) M, with a working pH range from 6.0 to 8.0 and a high selectivity. The detection limit is 5 x 10(-9) M. The response time for Cu2+ is less than 5 min with concentrations lower than 5 x 10(-6) M. The optode can be regenerated using 0.3 M EDTA (pH 9) and acetate buffer solution. The effect of the composition of the sensor membrane was studied, and the experimental conditions were optimized. The sensor has been used for direct determination of Cu2+ in water samples with satisfied results.

Platinum nanoparticles-doped sol-gel/carbon nanotubes composite electrochemical sensors and biosensors.

Platinum nanoparticle-doped sol-gel solution is prepared and used as a binder for multi-walled carbon nanotubes (CNT) for the fabrication of electrochemical sensors. Amine group containing sol-gel solution is selected to utilize the affinity of -NH(2) groups toward metal nanoparticles for stabilization the nanoparticles in solution. The resulting CNT-silicate material brings new capabilities for electrochemical devices by using the synergistic action of the electrocatalytic activity of Pt nanoparticles and CNT. The combined electrocatalytic activity permits low-potential detection of hydrogen peroxide with remarkably improved sensitivity. With the incorporation of glucose oxidase within the Pt-CNT-silicate matrix, a Pt-CNT paste-based biosensor has been constructed that responds more sensitively to glucose than CNT-based biosensor. The influences of the composite of the sol-gel solution, the quantity of the solution and the Pt nanoparticles loading are examined. In pH 6.98 phosphate buffer, almost interference free determination of glucose is realized at 0.1 V versus SCE with a linear range from 1 to 25 mM, a response time <15s, and the sensitivity is 0.98 microA mM(-1)cm(-2). The sensitivity of the Pt-CNT paste-based biosensor is almost four times larger than that of the CNT-based biosensor (0.27 microA mM(-1)cm(-2) at 0.1 V). The improved electrocatalytic activity and surface renewability made the Pt-CNT-silicate system a potential platform to immobilize different enzymes for other bioelectrochemical applications.

Carbon nanotube/cobalt hexacyanoferrate nanoparticle-biopolymer system for the fabrication of biosensors.

Cobalt hexacyanoferrate nanoparticles (CoNP) can be easily prepared by mixing hexacyanoferrate and cobalt chloride solution at room temperature. The nanoparticles were solubilized in aqueous solution of a biopolymer chitosan (CHIT). With the introduction of carbon nanotubes (CNT), the CoNP-CNT-CHIT system formed shows synergy between CNT and CoNP with the significant improvement of redox activity of CoNP due to the excellent electron-transfer ability of CNT. The CoNP-CNT-CHIT film modified glassy carbon electrode allows low potential detection of hydrogen peroxide with high sensitivity and fast response time. In particular, with the introduction of CNT, it amplified the H2O2 sensitivity by approximately 70 times compared to film of CoNP-CHIT. With the immobilization of glucose oxidase onto the electrode surface using glutaric dialdehyde, a biosensor that responds sensitively to glucose has been constructed. In pH 6.98 phosphate buffer, interference free determination of glucose has been realized at -0.2V versus saturated calomel electrode (SCE) with a linear range from 0.01 to 10 mM and response time<10s. The detection limit was 5 microM glucose (S/N=3).

Quartz crystal microbalance bioaffinity sensor for biotin based on mixed self-assembled monolayers and metastable molecular complex receptor.

A quartz crystal microbalance (QCM) sensor was proposed for the detection of small molecule biotin based on the mixed self-assembled monolayer (SAM) of thiols on gold substrate and the bioaffinity difference between an analyte (biotin) and an analogue compound (HABA) in binding avidin. Avidin formed a metastable complex with 2-[(4-hydroxyphenyl)azo]benzoic acid (HABA) immobilized on the crystal surface. When the sensor contacts a sample solution containing biotin, the avidin was released from the sensor surface to form a more stable complex with biotin in solution. The frequency change recorded is proportional to the desorbed mass of avidin, and there is a clear mathematic relationship between the frequency change and the biotin concentration. The use of mixed SAMs allows the stable attachment of bioreceptor molecules on the QCM, and enhances the amount of the immobilized molecules on the QCM, as a longer "space arm" in the mixed SAMs makes this monolayer membrane more accessible to capture the immobilized molecules. The proposed bioaffinity sensor has nice response to biotin in the range of 0.017-1.67 microg/mL. The sensor could be regenerated under very mild conditions simply by reimmersion of the sensor into a biotin solution to desorb the surplus avidin.

A highly selective potentiometric sensor of molybdate based on a mu-oxo-bridged manganeseporphyrin dimer.

A novel high-selective potentiometric sensor for molybdate was prepared with a PVC membrane combining mu-oxo-bis[5,10,15,20-tetra(p-methylphenyl)porphinatomanganese(III)] [[Mn(p-Me)TPP](2)O] as an electroactive material and 2-nitrophenyl octyl ether (o-NPOE) as a plasticizer in the percentage ratio of 3:65:32, [Mn(p-Me)TPP](2)O:o-NPOE:PVC (w:w). The sensor exhibited a linear response with a Nernstian slope of 30.5 mV per decade within a concentration range of 2.1 x 10(-6) to 1.0 x 10(-1) M MoO4(2-), with a working pH range from 5.0 to 12.5, and a fast response time of less than 15 s. The electrode showed improved selectivity toward molybdate with respect to common coexisting anions compared to monometalloporphyrin counterparts. Several electroactive materials and solvent mediators were compared and the experimental conditions were optimized. The sensor is preliminary applied to the assay of MoO4(2-) in corrosion inhibitor samples with satisfactory results.

MnO2-induced synthesis of fluorescent polydopamine nanoparticles for reduced glutathione sensing in human whole blood.

Polydopamine (PDA) nanoparticles, as a kind of popular polymer material, have attracted a great deal of attention from various areas including materials science, biomedicine, energy, environmental science and so on owing to their striking physicochemical properties. Herein, we reported for the first time the synthesis of intrinsic fluorescent PDA nanoparticles using MnO2 as an oxidant. In the presence of MnO2, dopamine was quickly oxidized into its quinone derivative, and autopolymerized into fluorescent PDA nanoparticles. Using fluorescent PDA nanoparticles as a fluorescence signal indicator, we further established a cost-effective sensor for rapid, sensitive and selective sensing of reduced glutathione (GSH) based on the redox reaction between MnO2 and GSH, and the key role of MnO2 in the formation of fluorescent PDA nanoparticles. GSH has the capability of reducing MnO2 into Mn(2+), which inhibited the formation of the fluorescent PDA nanoparticles. Thus, the concentration of GSH was directly related to the decreased fluorescence signal intensity of the PDA nanoparticles. The sensor showed good sensing performance for GSH detection with high sensitivity and desirable selectivity over other potential interfering species. Additionally, the sensor exhibited excellent practical applications for GSH detection in human whole blood samples, which presents potential applications in biological detection and clinical diagnosis.