Effect of Interfacial Action on the Generation and Transformation of Reactive Oxygen Species in Tripolyphosphate-Enhanced Heterogeneous Fe3O4/O2 Systems.

It has been reported that tripolyphosphate (TPP) can enhance the oxygenation of natural Fe(II)-containing minerals to produce reactive oxygen species (ROS). However, the molecular structure of the TPP-Fe(II) mineral surface complex and the role of this complex in the generation and transformation of ROS have not been fully characterized. In the present study, a heterogeneous magnetite (Fe3O4)/O2/TPP system was developed for the degradation of p-nitrophenol (PNP). The results showed that the addition of TPP significantly accelerated the removal of PNP in the Fe3O4/O2 system and extended the range of effective pH to neutral. Experiments combined with density functional theory calculations revealed that the activation of O2 mainly occurs on the surface of Fe3O4 induced by a structural Fe(II)-TPP complex, where the generated O2•- (intermediate active species) can be rapidly converted into H2O2, and then the •OH generated by the Fenton reaction is released into the solution. This increases the concentration of •OH produced and the efficiency of •OH produced relative to Fe(II) consumed, compared with the homogeneous system. Furthermore, the binding of TPP to the surface of Fe3O4 led to stretching and even cleavage of the Fe-O bonds. Consequently, more Fe(II)/(III) atoms are exposed to the solvation environment and are available for the binding of active O2 and O2•-. This study demonstrates how common iron minerals and O2 in the natural environment can be combined to yield a green remediation technology.

Mechanism of biochar functional groups in the catalytic reduction of tetrachloroethylene by sulfides.

In recent years, biochar has become of considerable interest for environmental applications, it can be used as a catalyst for sulfides reduction of perchloroethylene, but the crucial role of biochar properties played in catalyzing dechlorination remained ambiguous investigation. To pinpoint the critical functional groups, the modified biochars were respectively produced by HNO3, KOH and H2O2 with similar dimensional structures but different functional groups. Combined with the adsorption and catalytic results of different biochars, the acid-modified biochar had the best catalytic performance (99.9% removal) due to the outstanding specific surface area and ample functional groups. According to characterization and DFT results, carboxyl and pyridine nitrogen exhibited a positive correlation with the catalytic rate, indicating that their contribution to catalytic performance. Customizing biochar with specific functional groups removed depth demonstrated that the carboxyl was essential component. Further, alkaline condition was conducive to catalytic reduction, while tetrachloroethylene cannot be reduced under acidic conditions, because HS- and S2- mainly existed in alkaline environment and the sulfur-containing nucleophilic structure formed with biochar was more stable under this condition. Overall, this study opens new perspectives for in situ remediation by biochar in chlorinated olefin polluted anoxic environment and promotes our insight of modifying for biochar catalyst design.

Nanobiochar paper based electrochemical immunosensor for fast and ultrasensitive detection of microcystin-LR.

A portable, cheap and sensitive paper type electrochemical immunosensor was developed with conductive nanobiochar paper as the conductive layer and utilized for sensitive detection of microcystin-LR (MCLR) toxin in water. The paper immunosensor was constructed by coating of highly conductive and dispersible nanobiochar particle (nBC) and anti-MCLR antibody on the filter paper via dipping-drying method. The presence of MCLR could be specifically quantified amperometrically by the nBC-paper immunosensor with the response time of less than 5 min, and the lowest detection limit of 17 pM (0.017 µg/L) was achieved. Moreover, the proposed immunosensor exhibited high selectivity, reproducibility and storage stability, and was also used for environmental water detection with satisfactory recovery. The successful fabrication of low cost and ubiquitous biochar based paper type electrochemical immunosensing system would have significant value for the development of highly cost-effective electrochemical device.

Immobilization of laccase on magnetically separable biochar for highly efficient removal of bisphenol A in water.

Laccase was stably immobilized on a cost effective and nanosized magnetic biochar (L-MBC) by adsorption, precipitation and crosslinking, and it was used for high performance BPA removal. A large amount of enzyme could be immobilized on the magnetic biochar with high activity (2.251 U per mg MBC), and the L-MBC could be magnetically separated from the aqueous solution in 20 seconds. The successful immobilization of laccase was also confirmed via FTIR, SEM, and EDS analyses. The L-MBC presented better storage and stability performances, pH tolerance and thermal stability than the free laccase. It was found that BPA with an initial concentration of 25 mg L-1 could be thoroughly removed within 75 min, where BPA removal was attributed to enzymatic degradation and adsorption. In addition, the BPA removal efficiency by the L-MBC could be maintained above 85% even after seven cycles of repeated use. Due to high stability and efficient recyclability, the L-MBC-based biocatalyst has the potential to be a reliable method for treating BPA in environmental water sources.

High efficiency biotransformation of bisphenol A in a fluidized bed reactor using stabilized laccase in porous silica.

We developed a high efficiency bisphenol A (BPA) treating system in a fluidized bed reactor (FBR) using stabilized laccase (Lac) in mesoporous silica (SER/Lac). The SER/Lac, prepared by cross linking of each enzymes inside the porous silica using glutaraldehyde, presented improved stability than the free Lac over various pH and temperatures with shaking. When the SER/Lac was used for BPA biotransformation in a batch reaction, higher efficiency was achieved than the free or adsorbed Lac (ADS/Lac). Also, the SER/Lac presented better reusability compared to ADS/Lac, and it could be reused for three times without decrease of biotransformation efficiency, and half of it was remained after ten times use. Due to great stability and robustness property, SER/Lac was successfully applied for high efficiency and continuous BPA treatment in FBR with better performance than the batch reaction.

Electrochemical Enzyme Biosensor Bearing Biochar Nanoparticle as Signal Enhancer for Bisphenol A Detection in Water.

An electrochemical tyrosinase enzyme (Tyr) biosensor using a highly conductive sugarcane derived biochar nanoparticle (BCNP) as a transducer and signal enhancer (BCNPs/Tyr/Nafion/GCE) was developed for the sensitive detection of bisphenol A (BPA). The BCNPs/Tyr/Nafion/GCE biosensor exhibited improved amperometric current responses such as higher sensing signal, decreased impedance and lowered reduction potential compared with the Tyr/Nafion/GCE due to high conductivity property of the biochar nanoparticle. Under the optimized conditions, it could detect BPA in good sensitivity with linear range from 0.02 to 10 µM, and a lowest detection limit of 3.18 nM. Moreover, it showed a low Km value, high reproducibility and good selectivity over other reagents, and the BCNPs/Tyr complex solution also showed good stability with 86.9% of sensing signal maintained after one month storage. The biosensor was also successfully utilized for real water detection with high accuracy as validated by high performance liquid chromatography. Therefore, the biochar nanoparticle based enzyme biosensor proved to be a potential and reliable method for high performance detection of pollutants in the environment.

Multi-Functional Laccase Immobilized Hydrogel Microparticles for Efficient Removal of Bisphenol A.

Hghly stable, reusable, and multi-functional biocatalytic microparticles with Laccase (Lac) enzyme (Lac/particles) were synthesized for bisphenol A (BPA) removal from aqueous solution. The Lac/particles were prepared by encapsulating Lac enzymes into poly ethylene glycol (PEG) hydrogel via the UV assisted emulsion polymerization method followed by cross linking with glutaraldehyde (GA). The obtained Lac/particles were spherical and micron sized (137⁻535 µm), presenting high enzyme entrapment efficiency of 100%, high activity recovery of 18.9%, and great stability at various pHs (3⁻7) than the free Lac. The Lac/particles could adsorb the BPA into the catalytic particles in a short time, promoting contact between BPA and enzyme, and further enzymatically degrade them without the shaking process and independent surrounding buffer solution. The Lac/particles could be reused for another round BPA adsorption and biotranformation by maintaining over 90% of BPA removal efficiency after seven times reuse. The synergistic effects of adsorption and biocatalytical reaction of Lac/particles have significant values in high efficient and cost-effective BPA removal.

Graphite nanoparticle as nanoquencher for 17ß-estradiol detection using shortened aptamer sequence.

A graphite nanoparticle (GN) with a spherical shape and stacked by a few layered graphene sheets is an ideal fluorescent nanoquencher for the fluorophore. We developed a novel aptasensor based on fluorescence resonance energy transfer for the sensitive and specific detection of 17ß-estradiol (E2) by using the GN as a fluorescent nanoquencher and the shorter E2 specific aptamer as the sensing probe. The physiological and chemical properties of the aptasensor in response to the E2 capture were investigated with an atomic force microscopic analysis and an E2 detection principle was clarified accordingly. Also, it was demonstrated that the sensitivity of the aptasensor was affected by the length of aptamer and the particle size of the nanoquencher, and the highest sensitivity for E2 detection was achieved with a shorter aptamer of 35 base sequences and a smaller GN with a particle size of around 5 nm. And the detection limit was 1.02 ng mL-1. Moreover, this presented no cross reaction with E2 analogs and was successfully utilized for the real environmental water monitoring.

Direct Electrochemical Detection of Bisphenol A Using a Highly Conductive Graphite Nanoparticle Film Electrode.

We developed an accurate and sensitive sensor for electrochemical detection of bisphenol A (BPA) with a high-conductivity graphite nanoparticle (GN) film electrode. The GNs consisted of several stacked graphene sheets and showed a homogenous spherical shape, high conductivity, large surface area and good adsorption properties to BPA. The constructed GN film electrode exhibited improved amperometric current responses such as decreased impedance and lowered BPA oxidation potential compared with those of a pristine electrode, and also possessed a large surface area to allow fast electron transfer and BPA accumulation. A pre-accumulation process with BPA adsorption resulted in considerable current signal enhancement during BPA detection. The loading amount of GNs on the film electrode and the time for target BPA enrichment were optimized. The GN film electrode-based sensor showed high reproducibility and high selectivity for BPA over other reagents. Differential pulse voltammetry experiments revealed that the concentrations of BPA were linearly correlated with the current changes, and the lowest limit of detection of the sensor was 35 nM. Furthermore, the sensor showed great accuracy and reliability, as confirmed by high-performance liquid chromatography measurements. The sensor was also successfully used for BPA determination in groundwater samples, demonstrating its potential for real environmental analysis.

Enzyme incorporated microfluidic device for in-situ glucose detection in water-in-air microdroplets.

Droplet generating microfluidic systems can provide miniaturized bioanalytical tools by using the homogenous and high-throughput droplets as nanoreactors. In this study, we demonstrated a sensitive and in-situ glucose monitoring system using water-in-air droplets in an enzyme incorporated microfluidic device. A thin film structure of a glucose oxidase (GOx) enzyme immobilized hydrogel was constructed in the middle of the microfluidic channel, and nanoliter scaled water-in-air droplets which contain a glucose sample, horseradish peroxidase (HRP), and an Amplex Red substrate were generated by flow focusing of water phase with air. Once the droplets passed through the enzyme trapped hydrogel, the droplets temporarily halted and a GOx mediated catalytic reaction with glucose proceeded, resulting in producing fluorescent resorufin products in the droplets. With optimized conditions such as the thickness of a hydrogel film and the size and flowing rate of droplets, fluorescence intensities of the released droplets linearly increased in proportional to the glucose concentration up to 3mM, and the limit of detection was calculated as 6.64µM. A spiked glucose in a real urine sample was also successfully analyzed, and the functionality of the proposed enzyme immobilized microfluidic chip was maintained for at least two weeks without loss of enzymatic activity and detection sensitivity. Thus, our methodology suggests a novel droplet based glucose sensing chip which can monitor glucose in a real-time and high-throughput manner.

A highly sensitive immunoassay using antibody-conjugated spherical mesoporous silica with immobilized enzymes.

A highly sensitive immunoassay was developed by using antibody-conjugated spherical mesoporous silica with immobilized enzymes. The higher ratio of enzyme/antibody than conventional ELISA improved both the sensitivity and dynamic range. Especially, the use of spherical mesoporous silica could achieve a limit of detection (LOD) with a sensitivity that is 20 times more than that of ELISA using amorphous silica.

Circumferential alignment of vascular smooth muscle cells in a circular microfluidic channel.

The circumferential alignment of human aortic smooth muscle cells (HASMCs) in an orthogonally micropatterned circular microfluidic channel is reported to form an in vivo-like smooth muscle cell layer. To construct a biomimetic smooth muscle cell layer which is aligned perpendicular to the axis of blood vessel, a half-circular polydimethylsiloxane (PDMS) microchannel is first fabricated by soft lithography using a convex PDMS mold. Then, the orthogonally microwrinkle patterns are generated inside the half-circular microchannel by a strain responsive wrinkling method. During the UV treatment on a PDMS substrate with uniaxial 40% stretch and a subsequent strain releasing step, the microwrinkle patterns perpendicular to the axial direction of the circular microchannel are generated, which can guide the circumferential alignment of HASMCs during cultivation. The analysis of orientation angle, shape index, and contractile protein marker expression indicates that the cultured HASMCs reveal the in vivo-like cell phenotype. Finally, a fully circular microchannel is produced by bonding two half-circular microchannels, and the HASMCs are cultured circumferentially inside the channels with high alignment and viability for 5 days. These results demonstrated the creation of an in vivo-like 3D smooth muscle cell layer in the circular microfluidic channel which can provide a bioassay platforms for in-depth study of HASMC biology and vascular function.

Three-dimensional graphene micropillar based electrochemical sensor for phenol detection.

A three-dimensional (3D) graphene incorporated electrochemical sensor was constructed for sensitive enzyme based phenol detection. To form the 3D graphene structure, polydimethylsiloxane (PDMS) micropillars were fabricated in the microchannel by using a conventional photolithography and the surface was modified with 3-aminopropyltriethoxysilane. Then, the negatively charged graphene oxide sheets were electrostatically adsorbed on the PDMS micropillar surface, and reduced in the hydrazine vapor. The resultant 3D graphene film provides a conductive working electrode as well as an enzyme-mediated sensor with a large surface area. After bonded with an electrode patterned glass wafer, the 3D graphene based electrochemical sensor was produced. Using the 3D graphene as a working electrode, an excellent electron transfer property was demonstrated by cyclic voltammetry measurement in an electrolyte solution containing 1mM K3Fe(CN)6 and 0.1 M KCl. To utilize the 3D graphene as an enzyme sensor, tyrosinase enzymes were immobilized on the surface of the graphene micropillar, and the target phenol was injected in the microchannel. The enzyme catalytic reaction process was monitored by amperometric responses and the limit of detection for phenol was obtained as 50 nM, thereby suggesting that the 3D graphene micropillar structure enhances the enzyme biosensing capability not only by increasing the surface area for enzyme immobilization, but also by the superlative graphene conductivity property.

Fabrication of a circular PDMS microchannel for constructing a three-dimensional endothelial cell layer.

We describe a simple and efficient fabrication method for generating microfluidic channels with a circular cross-sectional geometry by exploiting the reflow phenomenon of a thick positive photoresist. Initial rectangular shaped positive photoresist micropatterns on a silicon wafer, which were fabricated by a conventional photolithography process, were converted into a half-circular shape by tuning the temperature to around 105 °C. Through optimization of the reflow conditions, we could obtain a perfect circular micropattern of the positive photoresist, and control the diameter in a range from 100 to 400 µm. The resultant convex half-circular photoresist was used as a template for fabricating a concave polydimethylsiloxane (PDMS) through a replica molding process, and a circular PDMS microchannel was produced by bonding two half-circular PDMS layers. A variety of channel dimensions and patterns can be easily prepared, including straight, S-curve, X-, Y-, and T-shapes to mimic an in vivo vascular network. To form an endothelial cell layer, we cultured primary human umbilical vein endothelial cells inside circular PDMS microchannels, and demonstrated successful cell adhesion, proliferation, and alignment along the channel.

A novel molecular beacon bearing a graphite nanoparticle as a nanoquencher for in situ mRNA detection in cancer cells.

Molecular beacons (MBs) have shown fascinating applications in many biological fields. However, exploration of cost-effective, sensitive, stable and efficient MB for in situ live cell- based assay has still room for improvement. In this regards, we have developed a novel MB which bears a spherical graphite nanoparticle (GN) as a fluorescent quencher. The GN resulted in the high quenching efficiency, and the presence of GN enhanced the biological stability and transfection of the MB into the cells, thereby enabling the real-time survivin mRNA detection and quantification in the MCF-7 breast cancer cells. These results demonstrated that the advancement of the proposed MB containing a GN nanoquencher can be used as a robust molecular probe for genetic analysis in the cells.

The photoluminescent graphene oxide serves as an acceptor rather than a donor in the fluorescence resonance energy transfer pair of Cy3.5-graphene oxide.

We have systematically studied the fluorescence resonance energy transfer (FRET) efficiency between the photoluminescent graphene oxide (GO) and Cy3.5 dye by controlling the donor-acceptor distance with a double stranded DNA and demonstrated that the GO serves as an acceptor rather than a donor in this FRET system.

Sensitive and high-fidelity electrochemical immunoassay using carbon nanotubes coated with enzymes and magnetic nanoparticles.

We demonstrate a highly sensitive electrochemical immunosensor based on the combined use of substrate recycling and carbon nanotubes (CNTs) coated with tyrosinase (TYR) and magnetic nanoparticles (MNP). Both TYR and MNP were immobilized on the surface of CNTs by covalent attachment, followed by additional cross-linking via glutaraldehyde treatment to construct multi-layered cross-linked TYR-MNP aggregates (M-EC-CNT). Magnetically capturable, highly active and stable M-EC-CNT were further conjugated with primary antibody against a target analyte of hIgG, and used for a sandwich-type immunoassay with a secondary antibody conjugated with alkaline phosphatase (ALP). In the presence of a target analyte, a sensing assembly of M-EC-CNT and ALP-conjugated antibody was attracted onto a gold electrode using a magnet. On an electrode, ALP-catalyzed hydrolysis of phenyl phosphate generated phenol, and successive TYR-catalyzed oxidation of phenol produced electrochemically measurable o-quinone that was converted to catechol in a scheme of substrate recycling. Combination of highly active M-EC-CNT and substrate recycling for the detection of hIgG resulted in a sensitivity of 27.6 nA ng(-1) mL(-1) and a detection limit of 0.19 ng mL(-1) (1.2 pM), respectively, representing better performance than any other electrochemical immunosensors relying on the substrate recycling with the TYR-ALP combination. The present immunosensing system also displayed a long-term stability by showing a negligible loss of electrochemical detection signal even after reagents were stored in an aqueous buffer at 4°C for more than 6 months.

Multiplexed immunoassay using the stabilized enzymes in mesoporous silica.

Multiplexed immunoassay system was developed using the enzyme-immobilized mesoporous silica in a form of nanoscale enzyme reactors (NERs), which improve the enzyme loading, activity, and stability. Glucose oxidase (GO) and trypsin (TR) were adsorbed into mesoporous silica and further crosslinked for the construction of NERs, and antibody-conjugated NERs were employed for the analysis of target antigens in a sandwich-type magnetic bead-based immunoassay. This approach, called as NER-LISA (NER-linked immunosorbent assay), generated signals out of enzyme reactions that correlated well with the concentration of target antigens. The detection limit of NER-LISA using NER-GO and anti-human IgG was 67pM human IgG, and the sensitivity was 20 times higher than that of the conventional ELISA using anti-human IgG conjugated GO. Antibody-conjugated NER-GO and NER-TR were successfully employed for the simultaneous detection of two target antigens (human IgG and chicken IgG) in a solution by taking advantage of signals at different wavelengths (absorbances at 570nm and 410nm, respectively) from the assays of GO and TR activities, demonstrating the potential of NER-LISA in multiplexed immunoassay. The NER-LISA approach also enabled the successful use of a protease (trypsin), because the NER approach can effectively retain the protease molecules within the mesoporous silica and prevent the digestion of antibodies and enzymes during the whole process of NER-LISA.

High performance immunoassay using immobilized enzyme in nanoporous carbon.

A highly stable immunoassay format was constructed using signal-generating enzyme immobilized in nanoporous carbon. A mesocellular carbon foam, called MSU-F-C, was loaded with horseradish peroxidase (HRP), followed by cross-linking of the enzyme using glutaraldehyde (GA) and modification of the surface with anti-human IgG through EDC/sulfo-NHS chemistry. The resulting MSU-F-C/HRP/anti-human IgG stably retained immobilized enzymes and antibodies, showing higher thermal stability. The MSU-F-C/HRP/anti-human IgG retained about 80 % of initial enzyme activity at 40 degrees C after a 5 h incubation, while the HRP/anti-human IgG conjugate resulted in almost 90% loss of initial activity in the same condition. In bead-based immunoassays, the signal amplification using MSU-F-C/HRP/anti-human IgG enabled the sensitive colorimetric detection of a target analyte, human IgG, in a detection limit of approximately 33 pM, with negligible cross-reactivity against rabbit and chicken IgGs.

Efficient enrichment and desalting of protein digests using magnetic mesocellular carbon foams in matrix-assisted laser desorption/ionization mass spectrometry.

We demonstrate that magnetic mesocellular carbon foams (Mag-MCF-C) can be effectively used for enrichment and desalting of protein digests or peptides in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The large mesocellular pores and surface area of Mag-MCF-C are likely to mainly contribute to high efficiency in enrichment and desalting of protein digests. The magnetic property of Mag-MCF-C enabled easy and simple enrichment and desalting process comprising adsorption, washing, and separation steps by using an external magnet. Following elution from Mag-MCF-C by using a matrix solution (CHCA in 70% ACN/0.1% TFA), the peptides were subjected to MALDI-MS analysis. As a result, MALDI mass spectra of peptides or tryptic protein digests were distinct even at a peptide concentration as low as 50 pM. The use of Mag-MCF-C resulted in significantly improved sequence coverage for protein identification when compared to other conventional methods. Mag-MCF-C will find applications in mass spectrometric analysis of low abundance peptides or protein digests with high sensitivity.