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A carbon fiber ionization (CFI) technique was developed for the mass spectrometric analysis of various organic compounds with different polarities. The design of the CFI technique was based on the good compatibility and dispersion of samples and solutions in different solvents on carbon fiber. As a fast, convenient, and versatile ionization method, CFI-MS is especially efficient for analyzing many low/nonpolar organic compounds, such as polycyclic aromatic hydrocarbons, long-chain aliphatic aldehydes, sensitive steroids, terpenoids, and organometallic compounds. Some of these compounds may not be well-analyzed by electrospray ionization or electron ionization mass spectrometry. On the basis of our experimental results, the major ion formation mechanism of CFI-MS was suggested to involve desorption in a steam-distillation-like process, and then, ionization occurred mainly via corona discharge under high voltage. CFI-MS could not only work alone but also be coupled with separation techniques. It works well when coupled with supercritical fluid chromatography (SFC) as well as in the analysis of exhaled human air. The high flexibility and versatility of CFI-MS has extended its applications in many areas, such as fast chemical screening, clinical testing, and forensic analysis. [ABSTRACT FROM AUTHOR]

Electrospray ionization (ESI) is a powerful ionization technique with a wide range of applications. However, the analytes in low/nonpolar solvents cannot be analyzed directly in electrospray ionization-mass spectrometry (ESI-MS), because low/nonpolar solvents are incompatible with ESI, because of their low conductivity. To circumvent this problem, we introduce an electrospray-based ionization method termed solvent-assisted electrospray ionization (SAESI). With the help of electrospray solvents at the tip of the spray needle, compounds in "non-electrospray ionization-friendly" solvents can be ionized directly using solvent-assisted electrospray ionization-mass spectrometry (SAESI-MS). The key features that the assistant solvent can be chosen flexibly and makes little interference to samples lead to better ionization performance in detection of organic reaction intermediates and real-time analysis of polymers and chiral drugs separated by gel permeation chromatography (GPC) and normal phase liquid chromatography (NPLC). Furthermore, it can achieve online hydrogen/deuterium (H/D) exchange reaction and even mitigate the signal suppression caused by strong acid modifiers in liquid chromatography. In addition, all parts of this device are commercially available and it only requires two parameters to be optimized, which makes SAESI easy to handle. [ABSTRACT FROM AUTHOR]

Dried blood spot (DBS) samples on filter paper are surging in popularity as a sampling and storage vehicle for a wide range of clinical and pharmaceutical applications. For example, a DBS sample is collected from every baby born in the province of Ontario, Canada, for quantification of approximately one hundred analytes that are used to screen for 28 conditions, including succinylacetone (SA), a marker for hepatorenal tyrosinemia. Unfortunately, the conventional methods used to evaluate DBS samples for newborn screening and other applications are tedious and slow, with limited options for automated analysis. In response to this challenge, we have developed a method to couple digital microfluidics (DMF) to nanoelectrospray ionization mass spectrometry (nESI-MS) for SA quantification in DBS samples. The new system is formed by sandwiching a pulled glass capillary emitter between the two DMF substrates such that the capillary emitter is immobilized without external seals or gaskets. Moreover, we introduce a new feedback control system that enables high-fidelity droplet manipulation across DBS samples without manual intervention. The system was validated by application to on-chip extraction, derivatization, and analysis of SA and other analytes from DBS samples, with comparable performance to gold-standard methods. We propose that the new methods described here can potentially contribute to a new generation of analytical techniques for quantifying analytes in DBS samples for a wide range of applications. [ABSTRACT FROM AUTHOR]

We introduce the marriage of two technologies: digital microfluidics (DMF), a technique in which droplets are manipulated by application of electrostatic forces on an array of electrodes coated by an insulator, and porous polymer monoliths (PPMs), a class of materials that is popular for use for solid-phase extraction and chromatography. In this work, circular PPM discs were formed in situ by dispensing and manipulating droplets of monomer solutions to designated spots on a DMF device followed by UV-initiated polymerization. We used PPM discs formed in this manner to develop a digital microfluidic solid-phase extraction (DMF-SPE) method, in which PPM discs are activated and equilibrated, samples are loaded, PPM discs are washed, and the samples are eluted, all using microliter droplets of samples and reagents. The new method has extraction efficiency (93%) comparable to that of pipet-based ZipTips and is compatible with preparative sample extraction and recovery for on-chip desalting, removal of surfactants, and preconcentration. We anticipate that DMF-SPE may be useful for a wide range of applications requiring preparative sample cleanup and concentration. [ABSTRACT FROM AUTHOR]

A selective, fast, and effective enzyme assay based on matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) for quantifying β-lactamase, an illegal additive in milk products, has been reported. The strengths of the mass spectrometric assay are its response to all substrate and products, simple and direct detection of the conversion of substrate, and facile determination of enzyme activity. Also, MALDI MS is tolerant to many buffer salts and reagents without the requirement of complicated sample pretreatment procedures. In this study, the approach was used to detect the presence of β-lactamases (BLA) in milk samples. The amount of BLA that could be determined in a milk sample is 6 x 10-3 U∙mL-1 by this approach. To test the strategy, it has been applied to the fortified milk (adding a BLA product known as an antimicrobial destroyer). It is then tested whether the pasteurization procedure of the milk process affects the activity of BI.A in milk samples. This study offers a perspective into the utility of MALDI-FTMS as an alternative detection tool for BLA screening in milk. [ABSTRACT FROM AUTHOR]

Digital microfluidics (DMF) is a fluid handling technique that enables manipulation of discrete droplets on an array of electrodes. There is considerable enthusiasm for this method because of the potential for array-based screening applications. A limitation for DMF is nonspecific adsorption of reagents to device surfaces. If a given device is used to actuate multiple reagents, this phenomenon can cause undesirable cross-contamination. A second limitation for DMF (and all other microfluidic systems) is the "world-to-chip" interface; it is notoriously difficult to deliver reagents and samples to such systems without compromising the oft-hyped advantages of rapid analyses and reduced reagent consumption. We introduce a new strategy for digital microfluidics, in which a removable plastic "skin" is used to (a) eliminate cross-contamination and (b) bridge the world-to-chip interface. We demonstrated the utility of this format by implementing on-chip protein digestion on immobilized enzyme depots. This new method has the potential to transform DMF from being a curiosity for aficionados into a technology that is useful for bio-chemical applications at large. [ABSTRACT FROM AUTHOR]

In this paper, we present a technique for the preparation of polymer nanowires with the protein molecule imprinted and binding sites at surface. These surface imprinting nanowires exhibit highly selective recognition for a variety of template proteins, including albumin, hemoglobin, and cytochrome c. This recognition may be through a multi-step adsorption, with the specificity conferred by hydrogen bonding and shape selectivity. Due to the protein imprinted sites are located at, or close to, the surface; these imprinted nanowires have a good site accessibility toward the target protein molecules. Furthermore, the large surface area of the nanowires results in large protein molecule binding capacity of the imprinted nanowires. [ABSTRACT FROM AUTHOR]

Presents correction to the article "Magnetite-Containing Spherical Silica Nanoparticles for Biocatalysis and Bioseparations" that was previously published in the December 2004 issue of the periodical "Analytical Chemistry."

Autofluorescence background in complex biological samples is a major challenge in achieving high sensitivity of fluorescence immunoassays (FIA). Here we report an X-ray luminescence-based immunoassay for high-sensitivity detection of biomarkers using X-ray scintillating nanotags. Due to the weak scattering and absorption of most biological chromophores by X-ray excitation, a low-dose X-ray source can be used to produce intense scintillating luminescence from the nanotags for autofluorescence-free biosensing. To demonstrate this concept, we designed and synthesized NaGdF4:Tb@NaYF4 core/shell nanoparticles as kind of high-efficiency X-ray scintillating nanotags, which are able to convert high-energy X-ray photons to visible light without autofluorescence in biological samples. Notably, strong X-ray absorption and minimized surface quenching arising from the heavy Gd3+/Tb3+ atoms and core/shell architecture of the nanoparticles were found to be critically important for high-efficiency X-ray excited luminescence for high-sensitivity biosensing. Our method allows for sensing alpha-fetoprotein (AFP) biomarkers with a detection limit down to 0.25 ng/mL. Moreover, the as-described X-ray luminescence immunoassay exhibited an excellent biological specificity, high stability, and sample recovery, implying an opportunity for applications in complex biological samples. Consequently, our method can be readily extended for multiplexing sensing and medical diagnosis. [ABSTRACT FROM AUTHOR]

An ultrasensitive and specific electrochemiluminescence (ECL) biosensor has been designed for the p53 DNA sequence, which is based on cascade signal amplification of nicking endonuclease assisted target recycling and hyperbranched rolling circle amplification (HRCA). First of all, biotin modified hairpin capture DNA (HP) probe was immobilized on the surface of streptavidin magnespheres paramagnetic particles (PMPs). Target DNA hybridized with the loop portion of the HP probe, therefore unfolding HP to form a double-stranded DNA (dsDNA) containing the specific nicking site of the nicking endonuclease. Then, the nicking endonuclease recognized the specific nicking site and cleaved the HP into two pieces, liberating target DNA and the complementary sequence piece for the padlock probe. The intact target DNA would initiate the next cycle of hybridization and cleavage, thereby releasing multiple complementary sequences for the padlock probes. The liberated complementary sequences hybridized with the padlock probes, subsequently inducing the HRCA reaction and generating numerous dsDNA segments. Herein, Ru(phen)32+ was embedded into dsDNA and worked as ECL signal reporter. The reaction products were eventually pretreated by dialysis tube with the cutoff membrane to remove the residual Ru(phen)32+ in the solution for the following ECL measurements. Using this cascade amplification strategy, an ultrasensitive p53 DNA sequence detection method was developed with a wide linear range from 0.05 to 100 fM and a low detection limit of 0.02 fM. Moreover, this cascade amplified ECL biosensor had specific recognition capacity for noncomplementary and single- and double-base mismatched DNA. The proposed ECL biosensor might have a great potential in biomedical research and clinic analysis. [ABSTRACT FROM AUTHOR]

A simple, sensitive, and selective immobilization-free electrochemical aptasensor had been developed which combines the advantages of the discrimination of the aggregation of long and short DNA on a negatively charged indium tin oxide (ITO) electrode, high selectivity of the aptamer, and high efficiency of exonuclease-catalyzed target recycling amplification. Ochratoxin A (OTA), a type of mycotoxin, has been chosen as the model target. Methylene blue (MB) labeled probe DNA had been hybridized with the OTA aptamer first, which cannot diffuse freely to the negative charged ITO electrode surface due to the repulsion of the negative charges, since the hybridized DNA contains large negative charges. In the presence of target (OTA), the aptamer prefers to form an OTA--aptamer complex in lieu of an aptamer--DNA duplex, which results in the dissociation of probe DNA from the probe DNA--aptamer complex The released probe DNA could be digested into mononucleotides, including a MB-labeled electroactive mononucleotide (eT), due to the employment of the Recjf exonuclease, a single-stranded DNA specific exonuclease. Since the eT contains little negative charge, it can difiuse easily to the negative charged ITO electrode surface, which results in the enhanced electrochemical response detected. At the same time, the aptamer in the OTA--aptamer complex can be digested by Recjf exonuclease also to liberate the target, which can participate in the next reaction cycling and realize the electrochemical signal amplification. Based on this strategy, an ultrasensitive homogeneous immobilization-free electrochemical aptasensor for OTA can be developed with a low detection limit (LOD) of 0.004 ng mL-1 (S/N = 3). The proposed biosensor combines the advantages of the simplicity of immobilization-free homogeneous ITO based electrochemical determination, high efficiency of exonuclease-catalyzed target recycling, and high selectivity of the aptamer. The fabricated biosensor has been applied to detect OTA in real samples with satisfactory results. The same strategy can be applied to develop biosensors for diverse targets. [ABSTRACT FROM AUTHOR]

Traditional electrochemical DNA biosensors need DNA immobilization on the electrode surface, which is tedious and time-consuming. In this study, a simple but ultraselective electrochemical DNA biosensor had been designed to determine target DNA species related to oral cancer overexpressed 1 in saliva, which combines the signal amplification of nicking endonuclease assisted target recycling with the immobilization-free electrochemical method. The complementary substrate strand of target DNA species contains a simple asymmetric sequence had been modified with a methylene blue at the 3' terminal first, which cannot diffuse easily to the negative charged ITO electrode surface due to the abundant negative charges. The presence of the target DNA would trigger the formation of double-stranded DNA (dsDNA). Then the nicking endonuclease can recognize the simple asymmetric sequence in the dsDNA and cleave the substrate strand of ds-DNA into two pieces, a long ssDNA and a 2-base ssDNA linked with methylene blue. The short one can diffuse easily to the negative charged ITO electrode surface and results in the enhanced electrochemical response detected. At the same time, the target DNA can dissociate from the dsDNA and trigger the next round of hybridization, cleavage, and releasing, which results in the signal amplification. This homogeneous DNA biosensor can detect as low as 0.35 pM (S/N = 3) target DNA. Compared with the traditional heterogeneous electrochemical DNA biosensors, which are tedious and time-consuming due to the complex DNA immobilization process, the assay not only owns the merits of simple and high efficiency since performed in a homogeneous solution but also exhibits a high distinction ability to single-base mismatch, double-bases mismatch, and noncomplementary DNA sequence. [ABSTRACT FROM AUTHOR]

Herein, a novel fluorescence sensor based on g-C3N4 nanosheet-MnO2 sandwich nanocomposite has been developed for rapid and selective sensing of glutathione (GSH) in aqueous solutions, as well as living cells. The graphitic-phase C3N4 (g-C3N4) nanosheet used here is a new type of carbon-based nanomaterial with high fluorescence quantum yield and high specific surface area. We demonstrate a facile one-step approach for the synthesis of a g-C3N4 nanosheet-MnO2 sandwich nanocomposite for the first time. The fluorescence of g-C3N4 nanosheet in this nanocomposite is quenched, which attributing to fluorescence resonance energy transfer (FRET) from a g-C3N4 nanosheet to the deposited MnO2. Upon the addition of GSH, MnO2 is reduced to Mn2+, which leads to the elimination of FRET. As a result, the fluorescence of g-C3N4 nanosheet is restored. Importantly, the chemical response of the g-C3N4-MnO2 nanocomposite exhibits great selectivity toward GSH relative to other electrolytes and biomolecules. Under the optimal conditions, the detection limit of 0.2 μM for GSH in aqueous solutions can be reached. Furthermore, the g-C3N4-MnO2 nanocomposite is confirmed to be membrane-permeable and have low cytotoxicity. Moreover, we successfully apply this sensor for visualizing and monitoring change of the intracellular GSH in living cells. Moreover, the proposed sensor shows satisfying performance, such as low cost, easy preparation, rapid detection, good bio compatibility, and turn-on fluorescence response. [ABSTRACT FROM AUTHOR]

Biosensors based on nanomaterials have been used for detection of various biological molecules with high sensitivity and selectivity. Herein, we developed a simple and ultrasensitive electrochemical DNA biosensor using long-range self-assembled DNA nanostructures as carriers for signal amplification, which can achieve an impressive detection limit of 5 aM human immunodeficiency virus (HIV) DNA even in complex biological samples. In this study, we designed two auxiliary probes. A cascade of hybridization events between the two auxiliary probes can lead to long-range self-assembly and form micrometer-long one-dimensional DNA nanostructures. In the presence of target DNA, each copy of the target can act as a trigger to connect a DNA nanostructure to a capture probe on the electrode surface. Then, a great amount of redox indicator [Ru(NH3)6]3+ can be electrostatically bound to the DNA nanostructures and eventually result in significantly amplified electrochemical signals. [ABSTRACT FROM AUTHOR]

In this work, we developed a simple and general method for highly sensitive detection of proteins and small molecules based on cyclic enzymatic signal amplification (CESA) and hairpin aptamer probe. Our detection system consists of a hairpin aptamer probe, a linker DNA, two sets of DNA-modified AuNPs, and nicking endonuclease (NEase). in the absence of a target, the hairpin aptamer probe and linker DNA can stably coexist in solution. Then, the linker DNA can assemble two sets of DNA-modified AuNPs, inducmg the aggregation of AuNPs. However, in the presence of a target, the hairpin structure of aptamer probe is opened upon interaction with the target to form an aptamer probe-target complex. Then, the probe-target complex can hybridize to the linker DNA. Upon formation of the duplex, the NEase recognizes specific nucleotide sequence and cleaves the linker DNA into two fragments. After nicking, the released probe-target complex can hybridize with another intact linker DNA and the cycle starts a new. The cleaved fragments of linker DNA are not able to assemble two sets of DNA-modified AuNPs, thus a red color of separated AuNPs can be I observed. Taking advantage of the AuNPs-based sensing technique, we are able to assay the target simply by UV-vis spectroscopy and even by the naked eye. Herein, we can detect the human thrombin with a detection limit of 50 pM and adenosine triphosphate (ATP) with a detection limit of 100 nM by the naked eye. This sensitivity is about 3 orders of magnitude higher than that of traditional AuNPs-based methods without amplification, in addition, this method is general since there is no requirement of the NEase recognition site in the aptamer sequence. Furthermore, we proved that the proposed method is capable of detecting the target in complicated biological samples. [ABSTRACT FROM AUTHOR]

Peptide—protein interactions have critical roles in biology. Monitoring peptide—protein interactions plays an important role in investigating molecular recognition, screening drugs, and designing biosensors. In this paper, we develop a novel fluorescent approach to monitor peptide—protein interactions based on the assembly of pyrene-labeled peptide and graphene oxide (GO). The pyrene-labeled peptide is strongly adsorbed on the surface of GO via π-π interactions and hydrophobic interactions. As a result, the proximity of the GO to the pyrene moiety effectively quenches the fluorescence of pyrene. In the presence of target protein, the competitive binding of the target protein with GO for peptide results in the restoration of fluorescence signal. This signaling mechanism makes it possible to monitor the peptide—protein interactions in a homogeneous real-time format. [ABSTRACT FROM AUTHOR]

In this paper, we present a general protocol for the making of surface-imprinted core—shell nanoparticles via surface reversible addition-fragmentation chain—transfer (RAFT) polymerization using RAFT agent functionalized model silica nanoparticles as the chain-transfer agent. In this protocol, trichloro(4-chloromethylphenyl)silane was immobilized on the surface of SiO2 nanoparticles, forming chloromethylphenyl functionalized silica (silica-Cl). RAFT agent functionalized silica was subsequently produced by substitute reaction of silica-Cl with PhC(S)SMgBr. The grafting copolyinerization of 4-vinylpyridine and ethylene glycol dimethacrylate using surface RAFT polymerization and in the presence of 2,4-dichiorophenoxyacetic acid as the template led to the formation of surface-imprinted core—shell nanoparticles The resulting surface-imprinted core—shell nanoparticles bind the original template 2,4-D with an appreciable selectivity over structurally related compounds. The potential use of the surface-imprinted core—shell nanoparticles as the recognition element in the competitive fluorescent binding assay for 2,4-D was also demonstrated. [ABSTRACT FROM AUTHOR]