Small-Molecule Analysis Based on DNA Strand Displacement Using a Bacteriorhodopsin Photoelectric Transducer: Taking ATP as an Example.

A uniformly oriented purple membrane (PM) monolayer containing photoactive bacteriorhodopsin has recently been applied as a sensitive photoelectric transducer to assay color proteins and microbes quantitatively. This study extends its application to detecting small molecules, using adenosine triphosphate (ATP) as an example. A reverse detection method is used, which employs AuNPs labeling and specific DNA strand displacement. A PM monolayer-coated electrode is first covalently conjugated with an ATP-specific nucleic acid aptamer and then hybridized with another gold nanoparticle-labeled nucleic acid strand with a sequence that is partially complementary to the ATP aptamer, in order to significantly minimize the photocurrent that is generated by the PM. The resulting ATP-sensing chip restores its photocurrent production in the presence of ATP, and the photocurrent recovers more effectively as the ATP concentration increases. Direct and single-step ATP detection is achieved in 15 min, with detection limits of 5 nM and a dynamic range of 5 nM-0.1 mM. The sensing chip exhibits high selectivity against other ATP analogs and is satisfactorily stable in storage. The ATP-sensing chip is used to assay bacterial populations and achieves a detection limit for Bacillus subtilis and Escherichia coli of 102 and 103 CFU/mL, respectively. The demonstration shows that a variety of small molecules can be simultaneously quantified using PM-based biosensors.

Direct and Label-Free Determination of Human Glycated Hemoglobin Levels Using Bacteriorhodopsin as the Biosensor Transducer.

Glycated hemoglobin (HbA1c) levels are an important index for the diagnosis and long-term control of diabetes. This study is the first to use a direct and label-free photoelectric biosensor to determine HbA1c using bacteriorhodopsin-embedded purple membranes (PM) as a transducer. A biotinylated PM (b-PM) coated electrode that is layered with protein A-oriented antibodies against hemoglobin (Hb) readily captures non-glycated Hb (HbA0) and generates less photocurrent. The spectra of bacteriorhodopsin and Hb overlap so the photocurrent is reduced because of the partial absorption of the incident light by the captured Hb molecules. Two HbA0 and HbA1c aptasensors that are prepared by conjugating specific aptamers on b-PM coated electrodes single-step detect HbA0 and HbA1c in 15 min, without cross reactivity, with detection limits of ≤0.1 µg/mL and a dynamic range of 0.1-100 µg/mL. Both aptasensors exhibit high selectivity and long-term stability. For the clinical samples, HbA0 concentrations and HbA1c levels that are measured with aptasensors correlate well with total Hb concentrations and the HbA1c levels that are determined using standard methods (correlation gradient = 0.915 ± 0.004 and 0.981 ± 0.001, respectively). The use of these aptasensors for diabetes care is demonstrated.

Fibroblast Growth Factor 9 Suppresses Striatal Cell Death Dominantly Through ERK Signaling in Huntington's Disease.

BACKGROUND/AIMS: Huntington's disease (HD) is a heritable neurodegenerative disorder, and there is no cure for HD to date. A type of fibroblast growth factor (FGF), FGF9, has been reported to play prosurvival roles in other neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease. However, the effects of FGF9 on HD is still unknown. With many similarities in the cellular and pathological mechanisms that eventually cause cell death in neurodegenerative diseases, we hypothesize that FGF9 might provide neuroprotective functions in HD. METHODS: In this study, STHdhQ7/Q7 (WT) and STHdhQ111/Q111 (HD) striatal knock-in cell lines were used to evaluate the neuroprotective effects of FGF9. Cell proliferation, cell death and neuroprotective markers were determined via the MTT assay, propidium iodide staining and Western blotting, respectively. The signaling pathways regulated by FGF9 were demonstrated using Western blotting. Additionally, HD transgenic mouse models were used to further confirm the neuroprotective effects of FGF9 via ELISA, Western blotting and immunostaining. RESULTS: Results show that FGF9 not only enhances cell proliferation, but also alleviates cell death as cells under starvation stress. In addition, FGF9 significantly upregulates glial cell line-derived neurotrophic factor (GDNF) and an anti-apoptotic marker, Bcl-xL, and decreases the expression level of an apoptotic marker, cleaved caspase 3. Furthermore, FGF9 functions through ERK, AKT and JNK pathways. Especially, ERK pathway plays a critical role to influence the effects of FGF9 toward cell survival and GDNF production. CONCLUSIONS: These results not only show the neuroprotective effects of FGF9, but also clarify the critical mechanisms in HD cells, further providing an insight for the therapeutic potential of FGF9 in HD.

Versatile Protein-A Coated Photoelectric Immunosensors with a Purple-Membrane Monolayer Transducer Fabricated by Affinity-Immobilization on a Graphene-Oxide Complexed Linker and by Shear Flow.

Bacteriorhodopsin-embedded purple membranes (PM) have been demonstrated to be a sensitive photoelectric transducer for microbial detection. To efficiently prepare versatile BR-based immunosensors with protein A as antibody captures, a large, high-coverage, and uniformly oriented PM monolayer was fabricated on an electrode as an effective foundation for protein A conjugation through bis-NHS esters, by first affinity-coating biotinylated PM on an aminated surface using a complex of oxidized avidin and graphene oxide as the planar linker and then washing the coating with a shear flow. Three different polyclonal antibodies, each against Escherichia coli, Lactobacillus acidophilus, and Streptococcus mutans, respectively, were individually, effectively and readily adsorbed on the protein A coated electrodes, leading to selective and sensitive quantitative detection of their respective target cells in a single step without any labeling. A single-cell detection limit was achieved for the former two cells. AFM, photocurrent, and Raman analyses all displayed each fabricated layer as well as the captured bacteria, with AFM particularly revealing the formation of a massive continuous PM monolayer on aminated mica. The facile cell-membrane monolayer fabrication and membrane surface conjugation techniques disclosed in this study may be widely applied to the preparation of different biomembrane-based biosensors.

Facile isolation of purple membrane from Halobacterium salinarum via aqueous-two-phase system.

Purple membrane (PM) is a part of cytoplasmic membrane in certain extreme halophilic microorganisms belonging to Domain Archaea. It transduces light energy to generate proton gradient for ATP synthesis in the microorganisms. Bacteriorhodopsin (BR) is the only protein in PM responsible for the generation of proton gradient. Generally, PM was purified from Halobacterium salinarum via a tedious and lengthy sucrose density gradient ultracentrifugation (SGU). In this work, a facile method based on polyethyleneglycol (PEG)-phosphate aqueous-two- phase extraction system (ATPS) was employed to purify PM from cell lysate of H. salinarum. The results showed that PM could be completely recovered from the interface of PEG-phosphate ATPS with BR purity ca 94.1% as measured by UV-visible absorption spectra. In comparison with PM obtained by SGU, the PM isolated by ATPS could achieve the same level of purity and photocurrent activity (ca 177.2nA/µgBR/cm(2)) as analyzed by SDS-PAGE and photocurrent measurement, respectively. The easily scalable and straightforward ATPS procedure demonstrated that PM can be purified and recovered more cost-effectively with a significantly reduced operation time that should lead to broader range applications of PM possible.

Effect of an automated dispensing cabinet system on drug distribution effectiveness in a surgical unit.

The use of the automated dispensing cabinet (ADC) for drug distribution in hospitals has become increasingly common and has numerous benefits. This retrospective study assessed the effectiveness of an ADC that uses integrated information technology in the drug distribution process in a surgical unit as part of a smart medical process improvement project at Taichung Veterans General Hospital in 2019. The outcomes include medicine delivery time, working time of healthcare professionals, transportation manpower, dispensing errors, and satisfaction of nursing staff with the medication distribution process. After ADC implementation, the average waiting time of standing orders decreased significantly for both on and off duty periods (40.0 ± 27.6 to 3.0 ± 3.9 min, P < 0.001; 45.2 ± 25.8 to 2.9 ± 2.9 min, P < 0.001; respectively). Similar results were observed with immediate or temporary medication orders (54.4 ± 31.5 to 2.0 ± 3.0 min, P < 0.001; 64.0 ± 47.5 to 1.5 ± 1.8 min, P < 0.001; respectively). The average time spent by operation room and post-operation room (OR/POR) nurses on communicating with ward nurses for medication delivery to OR/POR was shortened by 46.9 ± 4.4 h per month, and the average time pharmacists spent on dispensing immediate or temporary medication orders was shortened by 5.6 ± 0.2 h per month. The satisfaction of nursing staff with the OR/POR drug delivery process was significantly improved after ADC implementation (3.2 ± 0.8 vs 4.2 ± 0.7, P < 0.001). Our results showed that ADC implementation in surgical units simplified drug delivery processes, shortened drug delivery time, improved drug delivery timeliness for surgical patients, decreased dispensing errors, and increased nursing staff satisfaction. In conclusion, the implementation of ADC was beneficial for surgical units. To the best of our knowledge, there have been no studies on a similar ADC system.

Peptide-Coated Bacteriorhodopsin-Based Photoelectric Biosensor for Detecting Rheumatoid Arthritis.

An effective early diagnosis is important for rheumatoid arthritis (RA) management. This study reveals a novel RA detection method using bacteriorhodopsin as a photoelectric transducer, a light-driven proton pump in purple membranes (PMs). It was devised by covalently conjugating a PM monolayer-coated electrode with a citrullinated-inter-alpha-trypsin inhibitor heavy chain 3 (ITIH3)542-556 peptide that recognizes the serum RA-associated autoantibodies. The direct serum coating decreased the photocurrents in the biosensor, with the reduction in the photocurrent caused by coating with an RA-patient serum that is significantly larger than that with a healthy-control serum (38.1% vs. 20.2%). The difference in the reduction in the photocurrent between those two serum groups widened after the serum-coated biosensor was further labeled with gold nanoparticle (AuNP)-conjugated anti-IgA (anti-IgA-AuNP) (53.6% vs. 30.6%). Both atomic force microscopic (AFM) and Raman analyses confirmed the sequential peptide, serum, and anti-IgA-AuNP coatings on the PM-coated substrates. The reductions in the photocurrent measured in both the serum and anti-IgA-AuNPs coating steps correlated well with the results using commercial enzyme-linked immunosorbent assay kits (Spearman rho = 0.805 and 0.787, respectively), with both a sensitivity and specificity close to 100% in both steps. It was shown that an RA diagnosis can be performed in either a single- or two-step mode using the developed biosensor.

Fibroblast Growth Factor 9 Stimulates Neuronal Length Through NF-kB Signaling in Striatal Cell Huntington's Disease Models.

Proper development of neuronal cells is important for brain functions, and impairment of neuronal development may lead to neuronal disorders, implying that improvement in neuronal development may be a therapeutic direction for these diseases. Huntington's disease (HD) is a neurodegenerative disease characterized by impairment of neuronal structures, ultimately leading to neuronal death and dysfunctions of the central nervous system. Based on previous studies, fibroblast growth factor 9 (FGF9) may provide neuroprotective functions in HD, and FGFs may enhance neuronal development and neurite outgrowth. However, whether FGF9 can provide neuronal protective functions through improvement of neuronal morphology in HD is still unclear. Here, we study the effects of FGF9 on neuronal length in HD and attempt to understand the related working mechanisms. Taking advantage of striatal cell lines from HD knock-in mice, we found that FGF9 increases total neuronal length and upregulates several structural and synaptic proteins under HD conditions. In addition, activation of nuclear factor kappa B (NF-kB) signaling by FGF9 was observed to be significant in HD cells, and blockage of NF-kB leads to suppression of these structural and synaptic proteins induced by FGF9, suggesting the involvement of NF-kB signaling in these effects of FGF9. Taken these results together, FGF9 may enhance total neuronal length through upregulation of NF-kB signaling, and this mechanism could serve as an important mechanism for neuroprotective functions of FGF9 in HD.

FGF9 induces neurite outgrowth upon ERK signaling in knock-in striatal Huntington's disease cells.

AIMS: Huntington's disease (HD) is a neurodegenerative disease that causes deficits in neurite outgrowth, which suggests that enhancement of neurite outgrowth is a potential direction by which to improve HD. Our previous publications showed that fibroblast growth factor 9 (FGF9) provides anti-apoptosis and anti-oxidative functions in striatal cell models of HD through the extracellular signal-regulated kinases (ERK) pathway, and FGF9 also stimulates cytoskeletons to enhance neurite outgrowth via nuclear factor kappa B (NF-kB) signaling. In this study, we further demonstrate the importance of the ERK pathway for the neurite outgrowth induced by FGF9 in HD striatal models. MATERIALS AND METHODS: FGF9 was treated with ERK (U0126) or NF-kB (BAY11-7082) inhibitors in STHdhQ7/Q7 and STHdhQ111/Q111 striatal knock-in cell lines to examine neurite outgrowth, cytoskeletal markers, and synaptic proteins via immunofluorescence staining and Western blotting. NF-kB activity was analyzed by NF-kB promoter reporter assay. KEY FINDINGS: Here, we show that suppression of ERK signaling significantly inhibits FGF9-induced neurite outgrowth, cytoskeletal markers, and synaptic proteins in HD striatal cells. In addition, we also show suppression of ERK signaling significantly decreases FGF9-induced NF-kB activation, whereas suppression of NF-kB does not decrease FGF9-induced ERK signaling. These results suggest that FGF9 activates ERK signaling first, stimulates NF-kB upregulation, and then enhances neurite outgrowth in HD striatal cells. SIGNIFICANCE: We elucidate the more detailed mechanisms of neurite outgrowth enhanced by FGF9 in these HD striatal cells. This study may provide insights into targeting neurite outgrowth for HD therapy.

A biotin-hydrogel-coated quartz crystal microbalance biosensor and applications in immunoassay and peptide-displaying cell detection.

A biotin-coated quartz crystal microbalance (QCM) chip was prepared by dip-coating a long-chain alkanethiol-modified crystal with precoupled dextran-biotin hydrogels. The resulting biotin chip was used to affinity-immobilize streptavidin (SAv) and was then further employed for various biosensor assays. First, the SAv chip allowed efficient on-line binding of biotinylated bovine serum albumin (bBSA), followed by a sensitive and specific response toward anti-bovine serum albumin (BSA) antibodies. Three consecutive immunoassays were reproducibly demonstrated with a single chip. The apparent binding kinetics with k(on)=5.9 microM(-1) h(-1), k(off)=10.1 h(-1), and K(D)=1.71 microM was readily resolved by fitting the real-time sensorgrams. Second, the capability of the SAv chip to selectively recognize recombinant Escherichia coli with flagella displaying an artificial SAv binding peptide, Strep-tag II, was demonstrated by QCM analysis and verified by scanning transmission electron microscope (STEM) image analysis with biotin-coated gold nanoparticles as the label. Finally, the affinity of the cell-displayed Strep-tag II peptide to surface-coated SAv, K(D)=6.8 x 10(8) CFU/ml, was resolved on-line using equilibrium binding kinetics by QCM. This study presents an easy, economical, and reliable method of preparing high-performance SAv-coated biotin chips with potential for application in real-time repetitive immunoassays, on-line binding kinetics studies, and high-affinity peptide screening.

Fibroblast growth factor 9 activates anti-oxidative functions of Nrf2 through ERK signalling in striatal cell models of Huntington's disease.

Huntington's disease (HD) is a heritable neurodegenerative disorder, and has been characterized as an increase of oxidative stress in brain regions. In our previous results, we showed fibroblast growth factor 9 (FGF9) provides neuroprotective functions to suppress cell death in HD striatal cells dominantly through ERK signalling. However, whether the working mechanism of FGF9 is related to anti-oxidative stress in HD is still unknown. In this study, STHdhQ7/Q7 (Q7) and STHdhQ111/Q111 (Q111) striatal knock-in cell lines were used to examine the neuroprotective effects of FGF9 against oxidative stress in HD. Results show that FGF9 alleviates oxidative stress induced by starvation in Q7 and Q111 cells. The treatment of FGF9 not only induces upregulation and activation of nuclear factor erythroid 2-like 2 (Nrf2), a critical transcription factor for anti-oxidative stress, but also further upregulates its downstream targets, such as superoxide dismutase 2, gamma-glutamylcysteine synthetase and glutathione reductase. Furthermore, blockage of the Nrf2 pathway abolishes the anti-oxidative functions of FGF9, and inhibition of ERK signalling reduces the activation of the FGF9-Nrf2 pathway, resulting in higher level of oxidative stress in HD cells. These results support the neuroprotective effects of FGF9 against oxidative stress through the ERK-Nrf2 pathway, and imply one of potential strategies for therapy of HD.

Hydrogel-coated streptavidin piezoelectric biosensors and applications to selective detection of Strep-tag displaying cells.

Two different hydrogel-coated streptavidin (SAv) piezoelectric chips were investigated. One was directly prepared by immobilizing SAv molecules covalently onto a dextran-modified crystal, and the other one was indirectly prepared by physically adsorbing SAv onto a biotin-linked dextran surface. The covalent preparation yielded 80% more SAv-binding and better subsequent adsorption of biotinylated bovine serum albumin (bBSA). Both chips displayed the best binding affinity with bBSA at pH 5.0 in a flow injection analysis and exhibited reproductive real-time response during layer-by-layer assembly of a bBSA and SAv multilayer film. In the multilayer assembly, approximately 3-7 SAv molecules were captured by each immobilized bBSA, and the estimated apparent KD values of the binding of flowing bBSA with surface SAv were 0.24 and 0.11 microM in the first two cycles of the covalently prepared chip, respectively. Two Escherichia coli cells, each flagellum-displaying Strep-tag I and Strep-tag II, respectively, were selectively detected by both kinds of SAv chips. These studies suggest the potential application of both chips in real-time screening SAv affinity ligands from a cell-display random peptide library.

Direct, label-free, selective, and sensitive microbial detection using a bacteriorhodopsin-based photoelectric immunosensor.

A photoelectric immunosensor using purple membranes (PM) as the transducer, which contains photoactive bacteriorhodopsin, is here first demonstrated for direct and label-free microbial detection. Biotinylated polyclonal antibodies against Escherichia coli were immobilized on a PM-coated electrode through further surface biotinylation and bridging avidin or NeutrAvidin. The photocurrent generated by the antibody-coated sensor was reduced after incubation with E. coli K-12 cultures, with the reduction level increased with the culture populations. The immunosensor prepared via NeutrAvidin exhibited much better selectivity than the one prepared via avidin, recognizing almost none of the tested Gram-positive bacteria. Cultures with populations ranging from 1 to 107CFU/10mL were detected in a single step without any preprocessing. Both AFM and Raman analysis confirmed the layer-by-layer fabrication of the antibody-coated substrates as well as the binding of microorganisms. By investigating the effect of illumination orientation and simulating the photocurrent responses with an equivalent circuit model containing a chemical capacitance, we suggest that the photocurrent reduction was primarily caused by the light-shielding effect of the captured bacteria. Using the current fabrication technique, versatile bacteriorhodopsin-based photoelectric immunosensors can be readily prepared to detect a wide variety of biological cells.

Expression and functional analysis of pituitary tumor transforming gene-1 [corrected] in uterine leiomyomas.

Pituitary tumor-transforming gene-1 (PTTG-1) is a novel protooncogene overexpressed in numerous cancer cell lines and cancers. In this study we elucidate the expression of PTTG-1 in uterine leiomyomas and its functional role in the development of this disease. By comparing 23 pairs of leiomyomas and matched pairs of myometria, we found that the expression of PTTG-1 is significantly elevated in leiomyoma. The expression of PTTG-1 is independent of the menstrual cycle and is not affected by ovarian hormones. In contrast, basic fibroblast growth factor (bFGF) time- and dose-dependently stimulates PTTG-1 expression, which results in increasing cell proliferation. Forced expression of PTTG-1 by transient transfection stimulates bFGF and VEGF expression as well as changes the expression pattern of cell cycle proteins. Western blotting analysis demonstrates that the expressions of PTTG-1, bFGF, and the cell proliferation marker, proliferating cell nuclear antigen, are positively correlated with each other, which supports the hypothesis that the positive feedback loop between PTTG-1 and bFGF increases leiomyoma cell proliferation. In summary, we have shown for the first time that PTTG-1 is up-regulated in human uterine leiomyomas and that the positive feedback loop between PTTG-1 and bFGF may be pivotal in the growth of leiomyoma cells.

Foretinib inhibits angiogenesis, lymphangiogenesis and tumor growth of pancreatic cancer in vivo by decreasing VEGFR-2/3 and TIE-2 signaling.

Foretinib, a multiple kinase inhibitor undergoing clinical trials, could suppress the activity of hepatocyte growth factor (HGF) receptor c-MET and vascular endothelial growth factor receptor-2 (VEGFR-2). In addition, Foretinib may inhibit two critical lymphangiogenic signaling receptors VEGFR-3 and TIE-2. However, the effect of Foretinib on lymphatic endothelial cells (LECs) in vitro and lymphangiogenesis in vivo is still unknown. We found Foretinib decreased basal- and HGF-induced c-MET activity at low concentrations. However, Foretinib only reduced the proliferation of pancreatic cancer cells at high concentration reflecting the intrinsic chemoresistance of pancreatic cancer cells. Foretinib inhibited VEGF-A, VEGF-C and Angiopoetin-2 (ANG-2)-stimulated tube formation and sprouting of LECs by reducing VEGFR-2, VEGFR-3 and TIE-2 activation and increased apoptosis of LECs. In xenograft animal study, Foretinib suppressed tumor growth by inhibiting proliferation, angiogenesis and lymphangiogenesis. Additionally, Foretinib inhibited angiogenesis and lymphangiogenesis more significantly and exhibited low detrimental effect in orthotopic animal study. Collectively, we suggested that Foretinib simultaneously inhibits cancer cells and LECs to reduce pancreatic tumor growth in vivo and demonstrated for the first time that Foretinib suppresses angiogenesis and lymphangiogenesis by blocking VEGFR-2/3 and TIE-2 signaling.

Fibroblast growth factor-9 is an endometrial stromal growth factor.

Fibroblast growth factor-9 (FGF-9) is an autocrine/paracrine growth factor considered to be important for the growth and survival of motorneurons and prostate. In this study, we found that FGF-9 was expressed at high levels in normal uterine endometrium, especially during the late proliferative phase, which is coincident with the rise of estradiol and the time of uterine endometrial proliferation. Using quantitative RT-PCR analysis, we found that FGF-9 mRNA was expressed primarily by endometrial stromal cells. High affinity receptors of FGF-9 were detected in both epithelial and stromal cells but with distinct patterns. FGFR2IIIc and FGFR3IIIc are abundant in endometrial stromal cell. FGFR2IIIb is mostly expressed in endometrial epithelial cells, whereas FGFR3IIIb is found in both epithelial and stromal cells. Treatment with FGF-9 induces endometrial stromal proliferation in a dose-dependent manner. Expression of FGF-9 in stromal cells was induced by 17beta-estradiol but not by progesterone. Furthermore, the administration of 17beta-estradiol stimulates endometrial stromal cell proliferation and that can be inhibited by cotreatment with anti-FGF-9 antibody. Herein we demonstrate, for the first time, that FGF-9 is an autocrine estromedin endometrial stromal growth factor that plays roles in cyclic proliferation of uterine endometrial stroma.

Expression and mitogenic effect of fibroblast growth factor-9 in human endometriotic implant is regulated by aberrant production of estrogen.

Fibroblast growth factor-9 (FGF-9) is a steroid-regulated mitogen and survival factor for nerve and mesenchymal cells. In the current study, we determined the expression pattern and functional roles of FGF-9 in the ectopic endometriotic lesions. We found that FGF-9 and its receptors were effectively expressed by ectopic endometriotic tissues. The expression of FGF-9 was greater in the early stage of endometriosis, compared with the severe stage, which is consistent with concentration of 17 beta-estradiol in the peritoneal fluid of women with endometriosis. In addition, expression of FGF-9 in ectopic endometriotic stromal cell was inhibited by treatment with ICI 182,870 indicating it is likely regulated by estrogen in an autocrine manner. Administration of 17 beta-estradiol induced FGF-9, FGF receptor 2IIIc, and FGF receptor 3IIIc expression in endometriotic stromal cells. Concordant with this result, treatment of endometriotic stromal cells with 4-hydroxyandrostenedione (an aromatase inhibitor) or ICI 182,870 inhibited their proliferation, and that was reversed by coadministration with 17 beta-estradiol or FGF-9. In conclusion, expression of FGF-9 in endometriotic stromal cells is associated with aberrant production of estrogen. The capability of proliferation possessed by endometriotic stromal cell during menstruation when ovarian 17 beta-estradiol is in the nadir may be mediated, at least in part, by autocrined estrogen-stimulated expression of FGF-9 and its receptors.

A metal-chelating piezoelectric sensor chip for direct detection and oriented immobilization of polyHis-tagged proteins.

A metal-chelating piezoelectric (PZ) chip for direct detection and controlled immobilization of polyHis-tagged proteins has been demonstrated. The chip was prepared by covalently binding a hydrogel matrix complex of oxidized dextran and nitrilotriacetic acid (NTA) ligand onto an activated alkanethiol-modified PZ crystal. The resulting chip effectively captured Ni2+ ions onto its NTA surface, as disclosed by the resonant frequency shift of the crystal and an X-ray photoelectron spectroscopy analysis. The real-time frequency analysis revealed that the bare NTA chip was nonfouling, regenerable, and highly reusable during continuous repetitive injections of ion solutions and binding proteins. In addition, the chip displayed good long-term reusability and storage stability. The individual binding studies of a polyHis-tagged glutathione-S-transferase and its native untagged form on various metal-charged chips revealed that Co2+, Cu2+, and Ni2+ ions each had different immobilization ability on the NTA surface, as well as their binding ability and selectivity with the tagged protein. As a result, the tagged protein immobilized on the Ni2+-charged chip can actively be bound with its antibody and substrate. Further, the quantitative analyses of the tagged protein in crude cell lysate with a single Ni2+-charged chip and of its substrate with a protein-coated chip were also successfully demonstrated. Therefore, this study initiates the possibilities of oriented, reversible, and universal immobilization of any polyHis-tagged protein and its functional study using a real-time PZ biosensor.

Production, IMAC purification, and molecular modeling of N-carbamoyl-D-amino acid amidohydrolase C-terminally fused with a six-his peptide.

A six-His peptide was genetically engineered to the C-terminus of Agrobacterium radiobacter N-carbamoyl-D-amino acid amidohydrolase monomer to facilitate the protein purification with immobilized metal affinity chromatography (IMAC). The fusion enzyme, named as DCaseH, was overexpressed in Escherichia coli and one-step IMAC-purified. The production study showed that DCaseH was optimally produced at 15 degrees C for 25 h by the induction of 0.05 mM IPTG. Both Co(2+)-chelated TANOL gels and Ni(2+)-chelated nitriloacetic acid agarose gels efficiently purified DCaseH, with the former yielding purer enzyme than the latter. Highly pure DCaseH was obtained in the former purification with the addition of 5 mM imidazole in the washing buffer, and the specific enzyme activity was increased more than 11-fold. Denaturing IMAC purification successfully purified DCaseH from inclusion bodies that were mostly composed of the overexpressed DCaseH, while the attempt to refold the purified enzyme by either dialysis or solid-state refolding was not achieved. The purified native enzyme was optimally active at pH 6.5 and 50 degrees C, and the presence of 10% glycerol increased the activity. The molecular modeling of dimeric DCaseH indicated that the six-His tags were freely exposed to the protein surface, resulting in the selective and effective IMAC purification of DCaseH.

Effect of graphene oxide on affinity-immobilization of purple membranes on solid supports.

The effect of graphene oxide (GO) on the surface fabrication of purple membranes (PM) containing photosensitive bacteriorhodopsin is first reported in this study. GO was initially modified with biotin and then coupled with oxidized avidin to generate a GO-avidin complex, which was subsequently used as a linker to immobilize biotinylated PM (b-PM) onto amine-functionalized supports. Indium-tin-oxide glass coated with the GO-avidin complex was more hydrophilic than the electrode coated only with oxidized avidin, and the successive b-PM adsorption yielded a 1.4-fold higher (410 nA/cm(2)) photoelectric activity. AFM analysis on mica revealed that the GO-avidin complex layer had less surface roughness and dissipation energy than the pure oxidized avidin linker layer. For subsequent b-PM fabrication, GO addition not only reduced the stacking of immobilized b-PM patches but also improved their interior compactness and surface smoothness. This study demonstrates a convenient way to introduce GO into PM fabrication technology to provide enhanced surface morphology and photoelectric activity.