Phage-based biosensors for detection of microbes and biomarkers

The detection of microbial species and communities has always been an area of great importance. Rapid methods for microbial detection are especially important in the medicine, pharmacological, and food industry. The lack of rapid and cost-effective detection methods creates a challenge to control possible epidemics. New pathogenic microbes can cause a worldwide outbreak of diseases, pandemics. The majority of currently used detection methods for pathogens have low sensitivity and specificity. Human microbiota has been demanding and mostly unexplored area before modern high-throughput methods were established. These methods have elucidated a lot of connections between different diseases and composition changes of intestinal microbiota. However, sequencing a large number of samples takes a great deal of time, work and it requires centralized facilities. For microbiota, there are no actual comprehensive and rapid sensors. Biosensors have a great potential to respond to the challenges of microbial detection. The small physical size of the equipment, ease of use and operability outside centralized hospitals are appealing characteristics of this emerging class of diagnostic devices In this thesis, the applicability of newly developed rapid biosensors were evaluated as a tool for the detection of urinary tract infection and biomarkers. The studied methods are not based on traditional immunoassay detection technology, which usually relies on detecting a single antigen from the sample. Instead, a sensitive long lifetime luminescent europium label was used with different modulating probes to nonspecifically interact with simulated samples, but also with hospital samples. Chemical probes, or phages as biological probes, were used successfully to provide multiparameter luminescence data —a fingerprint from each sample. Phage-based methods were designed to meet the challenges of rapid detection of a single bacterial species. The second phage-based study was based on lysogenic phages and tested with hospital samples the assay time was reduced. The proof-of-principle method showed sensitivity and specificity at the 90% mark when compared to the standard culture method. The method was further developed and applied to detect specific biomarkers in a controlled chemical environment and finally to classify lethal prostate cancer against non-lethal ones from urine samples. The assay demonstrated a statistically significant difference between the two groups (pBakteriofageihin perustuvat biosensorit mikrobien ja biomarkkereiden havaitsemisessa Lääketieteessä, lääkeaineiden puhtaassa valmistuksessa ja elintarviketeollisuudessa mikrobien nopea havaitseminen on ensisijaisen tärkeää. Nopeille ja edullisille havaitsemismenetelmille on tarvetta varsinkin epidemioiden hallitsemisessa. Uudet patogeeniset mikrobit voivat aiheuttaa pandemioita, jotka alkuvaiheessa etenevät hyvin nopeasti maanosasta toiseen. Tämän päivän havaitsemismenetelmillä on yleisesti haasteita herkkyydessä ja spesifisyydessä. Ihmisen mikrobiota on ollut pitkään haastava ja tutkimaton alue. Tämä johtuu yksinkertaisesti siitä, ettei tähän tutkimusalueeseen ollut riittäviä tutkimusmenetemiä. Nämä nykyaikaiset menetelmät ovat selventäneet paljon yhteyksiä eri sairauksien ja suolen mikrobiotan koostumuksen muutosten välillä. Koko mikrobiotan testaukseen ei ole käytännössä todellisia pikatestijärjestelmiä. Biosensoreilla on mahdollista vastata mikrobien havaitsemisen asettamiin haasteisiin. Pieni koko, helppokäyttöisyys ja käytettävyys kenttäolosuhteissa ovat houkuttelevia tekijöitä orastavalle diagnostiikkalaitteiden luokalle. Tässä väitöskirjassa arvioitiin erilaisten nopeiden biosensorien soveltuvuutta mikrobien ja biomarkkerien havaitsemisessa. Työssä tutkitut menetelmät eivät perustuneet perinteisiin immunomääritystekniikoihin, jotka perustuvat yleensä yksittäisen antigeenin havaitsemiseen näytteestä. Tämän sijasta käytettiin näytteen kemialliselle ympäristölle herkkää europium-leimaa, jota käytettiin ei-spesifisesti vuorovaikutuksessa sekä simuloidun näytteen että potilasnäytteiden kanssa. Bakteriofagit toimivat biologisina koettimina ja tuottivat moneen eri parametriin perustuvaa luminesenssidataa -sormenjäljen jokaisesta yksittäisestä näytteestä. Bakteriofageihin perustuvat menetelmät kehitettiin ensin yksittäisten bakteerilajien havaitsemiseen ensin simuloiduissa olosuhteissa ja tämä jälkeen potilasnäytteistä. Menetelmää sovellettiin pidemmälle tunnistamaan spesifisistä biomarkkeria säädellyssä kemiallisessa ympäristössä. Lopulta menetelmää käytettiin luokittelemaan tappavat eturauhassyöpänäytteet ei-tappavista näytteistä. Luokittelutestin avulla havainnollistettiin tilastollisesti merkittävä ero näiden kahden näytetyypin välillä (p <0.0014). Yhteenvetona nämä havainnot osoittavat sen, että lantanidi-leimaan ja bakteriofaageihin perustuvaa menetelmää voidaan käyttää spesifisesti biomarkkerin, tai yksittäisen bakteerilajin havaitsemisee

Applications of Cellular Components in Engineered Environments

Manipulation of cellular components in synthetic environment has attracted interest for applications ranging from sensors to nanelectronics and from catalysis to biomedical devices. In such applications, biological-based processes such as specific recognition and self-assembly direct the formation of hierarchical structures, all at minimum energetic costs, with high efficiency and reliability. Among such cellular machines, microtubules and kinesins have attracted special interest. Inside the cells, microtubules are giving structural integrity while serving as regular and uniform tracks for transport of vesicles or organelles. Kinesin uses a microtubule track in vivo to progress to specific locations with processive and coordinated steps, all under the transformation of the chemical cycle of adenosine triphosphate (ATP) into mechanical work. Manipulation of microtubules and kinesins in vitro has been implemented for the transport of synthetic cargos like beads, nanoparticles or quantum dots to specific locations for molecular detection or diagnosis. However, in such applications, individual separation of a microtubule or kinesin-based complex, or parallel and yet individual sustainability and attainability of such complexes, could not be achieved. Main challenges consisted of biological components susceptibility to experimental conditions or lack of stability and complexity of tasks to be achieved under ambient temperature and pressure conditions. This thesis presents novel concepts and implementations of such cellular components in engineered environments. From studying single molecule assembly forces using atomic force microscopy approaches, to synthesizing novel self-assembled hybrid materials combining advantages of organic and inorganic components, and from application of cellular components in nanoelectronics or as the next generation tools for single molecule printing, the work included offers viable solutions for emerging science and engineering concepts that promote natural-based processes for synthetic applications

Visual and Plasmon Resonance Absorption Sensor for Adenosine Triphosphate Based on the High Affinity between Phosphate and Zr(IV)

Zr(IV) can form phosphate and Zr(IV) (–PO32−–Zr4+–) complex owing to the high affinity between Zr(IV) with phosphate. Zr(IV) can induce the aggregation of gold nanoparticles (AuNPs), while adenosine triphosphate(ATP) can prevent Zr(IV)-induced aggregation of AuNPs. Herein, a visual and plasmon resonance absorption (PRA)sensor for ATP have been developed using AuNPs based on the high affinity between Zr(IV)with ATP. AuNPs get aggregated in the presence of certain concentrations of Zr(IV). After the addition of ATP, ATP reacts with Zr(IV) and prevents AuNPs from aggregation, enabling the detection of ATP. Because of the fast interaction of ATP with Zr(IV), ATP can be detected with a detection limit of 0.5 μM within 2 min by the naked eye. Moreover, ATP can be detected by the PRA technique with higher sensitivity. The A520nm/A650nm values in PRA spectra increase linearly with the concentrations of ATP from 0.1 μM to 15 μM (r = 0.9945) with a detection limit of 28 nM. The proposed visual and PRA sensor exhibit good selectivity against adenosine, adenosine monophosphate, guanosine triphosphate, cytidine triphosphate and uridine triphosphate. The recoveries for the analysis of ATP in synthetic samples range from 95.3% to 102.0%. Therefore, the proposed novel sensor for ATP is promising for real-time or on-site detection of ATP

Structural and functional characterisation of the protein inhibitor of activated STAT3 (PIAS3)

The signal transducer and activator of transcription (STAT) and protein inhibitor of STAT(PIAS) system represent an elegant regulatory mechanism of transcriptional control IN mammalian cytokine signalling. Abnormal activation of the system is associated with immune disorders and a large group of diverse tumours. PIAS3 is a multiple domain protein with distinct functions involved in regulation of cytokine-mediated gene activation pathways.Its over-expression significantly inhibits cell growth and renders cancer cells more sensitive to drugs. The objective of this study was to structurally and biochemically characterise the function of the PIAS3 protein using in silico, in vivo and in vitro analysis approaches.The conservation pattern of the PIAS protein family and critical conserved residues in the PINIT (Proline, Isoleucine, Asparagine, Isoleucine, Tyrosine) domain were identified. The PINIT domain model was generated based on the PINIT domain structure of yeast PIAS3 homologue Siz1 and structural determinants in the PIAS3-STAT3 interaction were evaluated.Guided by the in silico findings, in vivo analysis of the localisation of the PIAS3, mutantderivatives of PIAS3 (PIAS3-L97A, PIAS3-R99N, PIAS3-R99Q), PINIT and acidic domain was conducted. PIAS3 was completely localised in the nucleus while PIAS3 mutants appeared to exhibit diffuse cytoplasmic distribution. The PINIT domain was predominantly localised in the nucleus with some apparent perinuclear staining while the acidic domain exhibited a predominantly perinuclear staining pattern. Further analysis of the PINIT domain and the effect of the mutants on PIAS3-STAT3 interaction were assessed by in vitro analysis. Guided by in silico analysis, the PINIT domain and mutant derivatives of PINIT domain (PINIT-L97A, PINIT-R99N, and PINIT-R99Q) were heterologously expressed in Escherichia coli and subsequently purified using a combination of immobilized metal affinity and size exclusion based chromatography. The size and structural elements of the PINIT domain and its mutants were characterised. The 23 kDa PINIT domain was found to exist as a monomer in solution and its secondary structure was shown to consist of 66 % β-sheets by fourier transformed infrared spectroscopy consistent with the generated homology model.Using surface plasmonresonance spectroscopy (SPR) the PINIT domain was shown to bind to STAT3 in a specific concentration dependent manner. Recombinant PINIT-L97A,PINITR99N and PINIT-R99Q mutants, which exhibited similar structural integrity to the wildtype, were found to abrogate binding to STAT3. These findings suggest that these residues form part of a potential binding surface for stat3. In conclusion, this study has provided evidence that the PINIT domain is an important determinant of PIAS3 interaction with STAT3 and that the interaction is mediated by defined conserved residues directly involved in the PINITSTAT3 interaction

Structural and biophysical characterisation of PERK kinase towards understanding ER stress sensing and activation of the Unfolded Protein Response

The unfolded protein response (UPR) is a cellular mechanism that detects the accumulation of misfolded proteins within the endoplasmic reticulum (ER). In mammalian cells, the UPR is mediated by three ER-transmembrane proteins: PERK, IRE1 and ATF6. Early studies in the field provided evidence for the role of BiP, the major ER Hsp70 chaperone, in UPR activation by binding to the luminal domains of PERK, IRE1 and ATF6 and maintaining them in an inactive state. However the underlying mechanism of ER stress sensing and UPR activation is not yet understood. This thesis presents (i) the novel X-ray crystal structure of PERK luminal domain and (ii) a biochemical study of the unconventional interaction between BiP chaperone and the luminal domains of PERK and IRE1 in vitro. Firstly, the structure of PERK luminal domain was solved in two oligomeric states: dimers and tetramers. Compelling evidence is provided for a role of tetramer formation in directing downstream UPR signalling. Secondly, the unprecedented and unconventional direct binding of PERK and IRE1 luminal domains to BiP is demonstrated. The binding surface was mapped to the nucleotide-binding domain (NBD) of BiP. As such, this points away from a substrate-chaperone interaction and rather implies BiP as an explicit UPR signalling component. Upon binding to CH1 unfolded protein, BiP is released from PERK and IRE1 luminal domains. BiP dissociation from PERK and IRE1 is known to lead to their activation. Based on the work presented in this thesis a novel mechanism of ER stress sensing and UPR activation by PERK and IRE1 is proposed. BiP NBD normally interacts with the luminal domains of PERK and IRE1 and represses UPR signalling. During ER stress, binding of unfolded proteins to BiP’s substrate binding domain leads to the dissociation of BiP-luminal domain complexes. As such, the luminal domains are free to intertwine, mediated by an extended α-helix, and form active tetramers competent for cytoplasmic UPR signalling.Open Acces

Chromogenic and fluorogenic chemosensors and reagents for anions. A comprehensive review of the years 2010-2011

This review focuses on examples reported in the years 2010¿2011 dealing with the design of chromogenic and fluorogenic chemosensors or reagents for anions.Santos Figueroa, LE.; Moragues Pons, ME.; Climent Terol, E.; Agostini, A.; Martínez Mañez, R.; Sancenón Galarza, F. (2013). Chromogenic and fluorogenic chemosensors and reagents for anions. A comprehensive review of the years 2010-2011. Chemical Society Reviews. 42(8):3489-3613. doi:10.1039/C3CS35429FS34893613428Martínez-Máñez, R., & Sancenón, F. (2003). Fluorogenic and Chromogenic Chemosensors and Reagents for Anions. Chemical Reviews, 103(11), 4419-4476. doi:10.1021/cr010421eKatayev, E. A., Ustynyuk, Y. A., & Sessler, J. L. (2006). Receptors for tetrahedral oxyanions. Coordination Chemistry Reviews, 250(23-24), 3004-3037. doi:10.1016/j.ccr.2006.04.013Suksai, C., & Tuntulani, T. (2003). Chromogenic anion sensors. Chemical Society Reviews, 32(4), 192. doi:10.1039/b209598jKim, S. K., Lee, D. H., Hong, J.-I., & Yoon, J. (2009). Chemosensors for Pyrophosphate. Accounts of Chemical Research, 42(1), 23-31. doi:10.1021/ar800003fBeer, P. (2000). Electrochemical and optical sensing of anions by transition metal based receptors. Coordination Chemistry Reviews, 205(1), 131-155. doi:10.1016/s0010-8545(00)00237-xZhou, Y., Xu, Z., & Yoon, J. (2011). Fluorescent and colorimetric chemosensors for detection of nucleotides, FAD and NADH: highlighted research during 2004–2010. Chemical Society Reviews, 40(5), 2222. doi:10.1039/c0cs00169dGunnlaugsson, T., Glynn, M., Tocci (née Hussey), G. M., Kruger, P. E., & Pfeffer, F. M. (2006). Anion recognition and sensing in organic and aqueous media using luminescent and colorimetric sensors. Coordination Chemistry Reviews, 250(23-24), 3094-3117. doi:10.1016/j.ccr.2006.08.017Amendola, V., Esteban-Gómez, D., Fabbrizzi, L., & Licchelli, M. (2006). What Anions Do to N−H-Containing Receptors. Accounts of Chemical Research, 39(5), 343-353. doi:10.1021/ar050195lGunnlaugsson, T., Ali, H. D. P., Glynn, M., Kruger, P. E., Hussey, G. M., Pfeffer, F. M., … Tierney, J. (2005). Fluorescent Photoinduced Electron Transfer (PET) Sensors for Anions; From Design to Potential Application. Journal of Fluorescence, 15(3), 287-299. doi:10.1007/s10895-005-2627-yWiskur, S. L., Ait-Haddou, H., Lavigne, J. J., & Anslyn, E. V. (2001). Teaching Old Indicators New Tricks. Accounts of Chemical Research, 34(12), 963-972. doi:10.1021/ar9600796Nguyen, B. T., & Anslyn, E. V. (2006). Indicator–displacement assays. Coordination Chemistry Reviews, 250(23-24), 3118-3127. doi:10.1016/j.ccr.2006.04.009Xu, Z., Chen, X., Kim, H. N., & Yoon, J. (2010). Sensors for the optical detection ofcyanide ion. Chem. Soc. Rev., 39(1), 127-137. doi:10.1039/b907368jKaur, K., Saini, R., Kumar, A., Luxami, V., Kaur, N., Singh, P., & Kumar, S. (2012). Chemodosimeters: An approach for detection and estimation of biologically and medically relevant metal ions, anions and thiols. Coordination Chemistry Reviews, 256(17-18), 1992-2028. doi:10.1016/j.ccr.2012.04.013Zhou, Y., & Yoon, J. (2012). Recent progress in fluorescent and colorimetric chemosensors for detection ofamino acids. Chem. Soc. Rev., 41(1), 52-67. doi:10.1039/c1cs15159bMoragues, M. E., Martínez-Máñez, R., & Sancenón, F. (2011). Chromogenic and fluorogenic chemosensors and reagents for anions. A comprehensive review of the year 2009. Chemical Society Reviews, 40(5), 2593. doi:10.1039/c0cs00015aAldrey, A., Núñez, C., García, V., Bastida, R., Lodeiro, C., & Macías, A. (2010). Anion sensing properties of new colorimetric chemosensors based on macrocyclic ligands bearing three nitrophenylurea groups. Tetrahedron, 66(47), 9223-9230. doi:10.1016/j.tet.2010.09.054Odago, M. O., Colabello, D. M., & Lees, A. J. (2010). A simple thiourea based colorimetric sensor for cyanide anion. Tetrahedron, 66(38), 7465-7471. doi:10.1016/j.tet.2010.07.006Piątek, P. (2011). A selective chromogenic chemosensor for carboxylate salt recognition. Chemical Communications, 47(16), 4745. doi:10.1039/c0cc05537aHe, X., Herranz, F., Cheng, E. C.-C., Vilar, R., & Yam, V. W.-W. (2010). Design, Synthesis, Photophysics, and Anion-Binding Studies of Bis(dicyclohexylphosphino)methane-Containing Dinuclear Gold(I) Thiolate Complexes with Urea Receptors. Chemistry - A European Journal, 16(30), 9123-9131. doi:10.1002/chem.201000647Lin, W.-C., Tseng, Y.-P., Lin, C.-Y., & Yen, Y.-P. (2011). Synthesis of alanine-based colorimetric sensors and enantioselective recognition of aspartate and malate anions. Organic & Biomolecular Chemistry, 9(15), 5547. doi:10.1039/c1ob05135kRegueiro-Figueroa, M., Djanashvili, K., Esteban-Gómez, D., de Blas, A., Platas-Iglesias, C., & Rodríguez-Blas, T. (2010). Towards Selective Recognition of Sialic Acid Through Simultaneous Binding to Its cis-Diol and Carboxylate Functions. European Journal of Organic Chemistry, 2010(17), 3237-3248. doi:10.1002/ejoc.201000186Carasel, I. A., Yamnitz, C. R., Winter, R. K., & Gokel, G. W. (2010). Halide Ions Complex and Deprotonate Dipicolinamides and Isophthalamides: Assessment by Mass Spectrometry and UV−Visible Spectroscopy. The Journal of Organic Chemistry, 75(23), 8112-8116. doi:10.1021/jo101749aRostami, A., Colin, A., Li, X. Y., Chudzinski, M. G., Lough, A. J., & Taylor, M. S. (2010). N,N′-Diarylsquaramides: General, High-Yielding Synthesis and Applications in Colorimetric Anion Sensing. The Journal of Organic Chemistry, 75(12), 3983-3992. doi:10.1021/jo100104gAmendola, V., Bergamaschi, G., Boiocchi, M., Fabbrizzi, L., & Milani, M. (2010). The Squaramide versus Urea Contest for Anion Recognition. Chemistry - A European Journal, 16(14), 4368-4380. doi:10.1002/chem.200903190Sola, A., Orenes, R. A., García, M. A., Claramunt, R. M., Alkorta, I., Elguero, J., … Molina, P. (2011). Unprecedented 1,3-Diaza[3]ferrocenophane Scaffold as Molecular Probe for Anions. Inorganic Chemistry, 50(9), 4212-4220. doi:10.1021/ic102314rLee, D. Y., Singh, N., Satyender, A., & Jang, D. O. (2011). An azo dye-coupled tripodal chromogenic sensor for cyanide. Tetrahedron Letters, 52(51), 6919-6922. doi:10.1016/j.tetlet.2011.10.061Haridas, V., Sahu, S., & Praveen Kumar, P. P. (2011). Triazole-based chromogenic and non-chromogenic receptors for halides. Tetrahedron Letters, 52(51), 6930-6934. doi:10.1016/j.tetlet.2011.10.066Park, J. J., Kim, Y.-H., Rhim, S., & Kang, J. (2012). Anion receptors with viologen molecular scaffold. Tetrahedron Letters, 53(2), 247-252. doi:10.1016/j.tetlet.2011.11.040Amendola, V., Fabbrizzi, L., Mosca, L., & Schmidtchen, F.-P. (2011). Urea-, Squaramide-, and Sulfonamide-Based Anion Receptors: A Thermodynamic Study. Chemistry - A European Journal, 17(21), 5972-5981. doi:10.1002/chem.201003411You, J.-M., Jeong, H., Seo, H., & Jeon, S. (2010). A new fluoride ion colorimetric sensor based on dipyrrolemethanes. Sensors and Actuators B: Chemical, 146(1), 160-164. doi:10.1016/j.snb.2010.02.042Farinha, A. S. F., Tomé, A. C., & Cavaleiro, J. A. S. (2010). (E)-3-(meso-Octamethylcalix[4]pyrrol-2-yl)propenal: a versatile precursor for calix[4]pyrrole-based chromogenic anion sensors. Tetrahedron Letters, 51(16), 2184-2187. doi:10.1016/j.tetlet.2010.02.091Lee, G. W., Kim, N.-K., & Jeong, K.-S. (2010). Synthesis of Biindole−Diazo Conjugates as a Colorimetric Anion Receptor. Organic Letters, 12(11), 2634-2637. doi:10.1021/ol100830bBose, P., & Ghosh, P. (2010). Visible and near-infrared sensing of fluoride by indole conjugated urea/thiourea ligands. Chemical Communications, 46(17), 2962. doi:10.1039/b919128cWang, L., He, X., Guo, Y., Xu, J., & Shao, S. (2011). Tris(indolyl)methene molecule as an anion receptor and colorimetric chemosensor: tunable selectivity and sensitivity for anions. Org. Biomol. Chem., 9(3), 752-757. doi:10.1039/c0ob00472cTetilla, M. A., Aragoni, M. C., Arca, M., Caltagirone, C., Bazzicalupi, C., Bencini, A., … Meli, V. (2011). Colorimetric response to anions by a «robust» copper(ii) complex of a [9]aneN3 pendant arm derivative: CN− and I− selective sensing. Chemical Communications, 47(13), 3805. doi:10.1039/c0cc04500dKundu, T., Mobin, S. M., & Lahiri, G. K. (2010). Paramagnetic ruthenium-biimidazole derivatives [(acac)2RuIII(LHn)]m, n/m = 2/+, 1/0, 0/−. Synthesis, structures, solution properties and anion receptor features in solution state. Dalton Transactions, 39(17), 4232. doi:10.1039/b919036hLee, C.-H., Lee, S., Yoon, H., & Jang, W.-D. (2011). Strong Binding Affinity of a Zinc-Porphyrin-Based Receptor for Halides through the Cooperative Effects of Quadruple CH Hydrogen Bonds and Axial Ligation. Chemistry - A European Journal, 17(49), 13898-13903. doi:10.1002/chem.201101884Swinburne, A. N., Paterson, M. J., Fischer, K. H., Dickson, S. J., Wallace, E. V. B., Belcher, W. J., … Steed, J. W. (2010). Colourimetric Carboxylate Anion Sensors Derived from Viologen-Based Receptors. Chemistry - A European Journal, 16(5), 1480-1492. doi:10.1002/chem.200902609Kannappan, R., Bucher, C., Saint-Aman, E., Moutet, J.-C., Milet, A., Oltean, M., … Chaix, C. (2010). Viologen-based redox-switchable anion-binding receptors. New Journal of Chemistry, 34(7), 1373. doi:10.1039/b9nj00757aKumari, N., Jha, S., & Bhattacharya, S. (2011). Colorimetric Probes Based on Anthraimidazolediones for Selective Sensing of Fluoride and Cyanide Ion via Intramolecular Charge Transfer. The Journal of Organic Chemistry, 76(20), 8215-8222. doi:10.1021/jo201290aAmendola, V., Boiocchi, M., Fabbrizzi, L., & Fusco, N. (2011). Putting the Anion into the Cage - Fluoride Inclusion in the Smallest Trisimidazolium Macrotricycle. European Journal of Organic Chemistry, 2011(32), 6434-6444. doi:10.1002/ejoc.201100902Kumar, A., Kumar, V., & Upadhyay, K. K. (2011). A ninhydrin based colorimetric molecular switch for Hg2+ and CH3COO−/F−. Tetrahedron Letters, 52(50), 6809-6813. doi:10.1016/j.tetlet.2011.10.046Bao, X., & Zhou, Y. (2010). Synthesis and recognition properties of a class of simple colorimetric anion chemosensors containing OH and CONH groups. Sensors and Actuators B: Chemical, 147(2), 434-441. doi:10.1016/j.snb.2010.03.068Lou, X., Zhang, Y., Li, Q., Qin, J., & Li, Z. (2011). A highly specific rhodamine-based colorimetric probe for hypochlorites: a new sensing strategy and real application in tap water. Chemical Communications, 47(11), 3189. doi:10.1039/c0cc04911eShang, X.-F., Su, H., Lin, H., & Lin, H.-K. (2010). A supramolecular optic sensor for selective recognition AMP. Inorganic Chemistry Communications, 13(8), 999-1003. doi:10.1016/j.inoche.2010.04.006Mendy, J. S., Saeed, M. A., Fronczek, F. R., Powell, D. R., & Hossain, M. A. (2010). Anion Recognition and Sensing by a New Macrocyclic Dinuclear Copper(II) Complex: A Selective Receptor for Iodide. Inorganic Chemistry, 49(16), 7223-7225. doi:10.1021/ic100686mMahato, P., Ghosh, A., Mishra, S. K., Shrivastav, A., Mishra, S., & Das, A. (2011). Zn(II)−Cyclam Based Chromogenic Sensors for Recognition of ATP in Aqueous Solution Under Physiological Conditions and Their Application as Viable Staining Agents for Microorganism. Inorganic Chemistry, 50(9), 4162-4170. doi:10.1021/ic200223gMahato, P., Ghosh, A., Mishra, S. K., Shrivastav, A., Mishra, S., & Das, A. (2010). Zn(II) based colorimetric sensor for ATP and its use as a viable staining agent in pure aqueous media of pH 7.2. Chemical Communications, 46(48), 9134. doi:10.1039/c0cc01996hDalla Cort, A., Forte, G., & Schiaffino, L. (2011). Anion Recognition in Water with Use of a Neutral Uranyl-salophen Receptor. The Journal of Organic Chemistry, 76(18), 7569-7572. doi:10.1021/jo201213eDas, P., Mandal, A. K., Kesharwani, M. K., Suresh, E., Ganguly, B., & Das, A. (2011). Receptor design and extraction of inorganic fluoride ion from aqueous medium. Chemical Communications, 47(26), 7398. doi:10.1039/c1cc11458aBaumes, L. A., Buaki, M., Jolly, J., Corma, A., & Garcia, H. (2011). Fluorimetric detection and discrimination of α-amino acids based on tricyclic basic dyes and cucurbiturils supramolecular assembly. Tetrahedron Letters, 52(13), 1418-1421. doi:10.1016/j.tetlet.2011.01.071Baumes, L. A., Buaki Sogo, M., Montes-Navajas, P., Corma, A., & Garcia, H. (2010). A Colorimetric Sensor Array for the Detection of the Date-Rape Drug γ-Hydroxybutyric Acid (GHB): A Supramolecular Approach. Chemistry - A European Journal, 16(15), 4489-4495. doi:10.1002/chem.200903127Chifotides, H. T., Schottel, B. L., & Dunbar, K. R. (2010). The π-Accepting Arene HAT(CN)6 as a Halide Receptor through Charge Transfer: Multisite Anion Interactions and Self-Assembly in Solution and the Solid State. Angewandte Chemie International Edition, 49(40), 7202-7207. doi:10.1002/anie.201001755Gu, X., Liu, C., Zhu, Y.-C., & Zhu, Y.-Z. (2011). Development of a boron-dipyrromethene-Cu2+ ensemble based colorimetric probe toward hydrogen sulfide in aqueous media. Tetrahedron Letters, 52(39), 5000-5003. doi:10.1016/j.tetlet.2011.07.004Männel-Croisé, C., Meister, C., & Zelder, F. (2010). «Naked-Eye» Screening of Metal-Based Chemosensors for Biologically Important Anions. Inorganic Chemistry, 49(22), 10220-10222. doi:10.1021/ic1015115Watchasit, S., Kaowliew, A., Suksai, C., Tuntulani, T., Ngeontae, W., & Pakawatchai, C. (2010). Selective detection of pyrophosphate by new tripodal amine calix[4]arene-based Cu(II) complexes using indicator displacement strategy. Tetrahedron Letters, 51(26), 3398-3402. doi:10.1016/j.tetlet.2010.04.095Mateus, P., Delgado, R., Brandão, P., & Félix, V. (2011). Recognition of Oxalate by a Copper(II) Polyaza Macrobicyclic Complex. Chemistry - A European Journal, 17(25), 7020-7031. doi:10.1002/chem.201100428Chen, Z., Lu, Y., He, Y., & Huang, X. (2010). Recognition of pyrophosphate anion in aqueous solution using the competition displacement method. Sensors and Actuators B: Chemical, 149(2), 407-412. doi:10.1016/j.snb.2010.06.038Müller-Graff, P.-K., Szelke, H., Severin, K., & Krämer, R. (2010). Pattern-based sensing of sulfated glycosaminoglycans with a dynamic mixture of iron complexes. Organic & Biomolecular Chemistry, 8(10), 2327. doi:10.1039/c000420kHu, Z.-Q., Wang, X.-M., Feng, Y.-C., Ding, L., Li, M., & Lin, C.-S. (2011). A novel colorimetric and fluorescent chemosensor for acetate ions in aqueous media based on a rhodamine 6G–phenylurea conjugate in the presence of Fe(iii) ions. Chem. Commun., 47(5), 1622-1624. doi:10.1039/c0cc04136jSingh, N., & Jang, D. O. (2011). A selective ATP chromogenic sensor for use in an indicator displacement assay. Tetrahedron Letters, 52(39), 5094-5097. doi:10.1016/j.tetlet.2011.07.096Ghosh, K., & Ranjan Sarkar, A. (2011). Pyridinium-based symmetrical diamides as chemosensors in visual sensing of citrate through indicator displacement assay (IDA) and gel formation. Organic & Biomolecular Chemistry, 9(19), 6551. doi:10.1039/c1ob05707cAtta, A. K., Ahn, I.-H., Hong, A.-Y., Heo, J., Kim, C. K., & Cho, D.-G. (2012). Fluoride indicator that functions in mixed aqueous media: hydrogen bonding effects. Tetrahedron Letters, 53(5), 575-578. doi:10.1016/j.tetlet.2011.11.099Perry-Feigenbaum, R., Sella, E., & Shabat, D. (2011). Autoinductive Exponential Signal Amplification: A Diagnostic Probe for Direct Detection of Fluoride. Chemistry - A European Journal, 17(43), 12123-12128. doi:10.1002/chem.201101796Rajamalli, P., & Prasad, E. (2011). Low Molecular Weight Fluorescent Organogel for Fluoride Ion Detection. Organic Letters, 13(14), 3714-3717. doi:10.1021/ol201325jBhaumik, C., Das, S., Maity, D., & Baitalik, S. (2011). A terpyridyl-imidazole (tpy-HImzPh3) based bifunctional receptor for multichannel detection of Fe2+ and F− ions. Dalton Transactions, 40(44), 11795. doi:10.1039/c1dt10965kIsaad, J., & Perwuelz, A. (2010). New color chemosensors for cyanide based on water soluble azo dyes. Tetrahedron Letters, 51(44), 5810-5814. doi:10.1016/j.tetlet.2010.08.098Wade, C. R., & Gabbaï, F. P. (2010). Cyanide Anion Binding by a Triarylborane at the Outer Rim of a Cyclometalated Ruthenium(II) Cationic Complex. Inorganic Chemistry, 49(2), 714-720. doi:10.1021/ic9020349Ábalos, T., Jiménez, D., Moragues, M., Royo, S., Martínez-Máñez, R., Sancenón, F., … Gil, S. (2010). Multi-channel receptors based on thiopyrylium functionalised with macrocyclic receptors for the recognition of transition metal cations and anions. Dalton Transactions, 39(14), 3449. doi:10.1039/b921486kÁbalos, T., Royo, S., Martínez-Máñez, R., Sancenón, F., Soto, J., Costero, A. M., … Parra, M. (2009). Surfactant-assisted chromogenic sensing of cyanide in water. New Journal of Chemistry, 33(8), 1641. doi:10.1039/b909705hSumiya, S., Doi, T., Shiraishi, Y., & Hirai, T. (2012). Colorimetric sensing of cyanide anion in aqueous media with a fluorescein–spiropyran conjugate. Tetrahedron, 68(2), 690-696. doi:10.1016/j.tet.2011.10.097Shiraishi, Y., Itoh, M., & Hirai, T. (2011). Rapid colorimetric sensing of cyanide anion in aqueous media with a spiropyran derivative containing a dinitrophenolate moiety. Tetrahedron Letters, 52(13), 1515-1519. doi:10.1016/j.tetlet.2011.01.110Shiraishi, Y., Itoh, M., & Hirai, T. (2011). Colorimetric response of spiropyran derivative for anions in aqueous or organic media. Tetrahedron, 67(5), 891-897. doi:10.1016/j.tet.2010.12.021Isaad, J., & Achari, A. E. (2011). Biosourced 3-formyl chromenyl-azo dye as Michael acceptor type of chemodosimeter for cyanide in aqueous environment. Tetrahedron, 67(31), 5678-5685. doi:10.1016/j.tet.2011.05.083Isaad, J., & El Achari, A. (2011). A novel cyanide chemodosimeter based on trifluoroacetamide benzhydrol-2 as binding motif: importance of substituent positioning on intra-molecular charge transfer. Tetrahedron, 67(23), 4196-4201. doi:10.1016/j.tet.2011.04.059Park, I. S., Heo, E.-J., & Kim, J.-M. (2011). A photochromic phenoxyquinone based cyanide ion sensor. Tetrahedron Letters, 52(19), 2454-2457. doi:10.1016/j.tetlet.2011.02.105Tang, X., Liu, W., Wu, J., Zhao, W., Zhang, H., & Wang, P. (2011). A colorimetric chemosensor for fast detection of thiols based on intramolecular charge transfer. Tetrahedron Letters, 52(40), 5136-5139. doi:10.1016/j.tetlet.2011.07.111Wei, W., Liang, X., Hu, G., Guo, Y., & Shao, S. (2011). A highly selective colorimetric probe based on 2,2′,2″-trisindolylmethene for cysteine/homocysteine. Tetrahedron Letters, 52(13), 1422-1425. doi:10.1016/j.tetlet.2010.07.182Cui, K., Zhang, D., Zhang, G., & Zhu, D. (2010). A highly selective naked-eye probe for hypochlorite with the p-methoxyphenol-substituted aniline compound. Tetrahedron Letters, 51(46), 6052-6055. doi:10.1016/j.tetlet.2010.09.041Kim, M. H., Kim, S., Jang, H. H., Yi, S., Seo, S. H., & Han, M. S. (2010). A gold nanoparticle-based colorimetric sensing ensemble for the colorimetric detection of cyanide ions in aqueous solution. Tetrahedron Letters, 51(36), 4712-4716. doi:10.1016/j.tetlet.2010.07.002Zhang, S., Wang, J., Han, L., Li, C., Wang, W., & Yuan, Z. (2010). Colorimetric detection of bis-phosphorylated peptides using zinc(ii) dipicolylamine-appended gold nanoparticles. Sensors and Actuators B: Chemical, 147(2), 687-690. doi:10.1016/j.snb.2010.03.071Feng, D.-Q., Liu, G., Zheng, W., Liu, J., Chen, T., & Li, D. (2011). A highly selective and sensitive on–off sensor for silver ions and cysteine by light scattering technique of DNA-functionalized gold nanoparticles. Chemical Communications, 47(30), 8557. doi:10.1039/c1cc12377gCao, R., & Li, B. (2011). A simple and sensitive method for visual detection of heparin using positively-charged gold nanoparticles as colorimetric probes. Chemical Communications, 47(10), 2865. doi:10.1039/c0cc05094fLiu, C.-Y., & Tseng, W.-L. (2011). Colorimetric assay for cyanide and cyanogenic glycoside using polysorbate 40-stabilized gold nanoparticles. Chemical Communications, 47(9), 2550. doi:10.1039/c0cc04591hZhang, M., Liu, Y.-Q., & Ye, B.-C. (2011). Rapid and sensitive colorimetric visualization of phthalates using UTP-modified gold nanoparticles cross-linked by copper(ii). Chemical Communications, 47(43), 11849. doi:10.1039/c1cc14772bLi, H., Li, F., Han, C., Cui, Z., Xie, G., & Zhang, A. (2010). Highly sensitive and selective tryptophan colorimetric sensor based on 4,4-bipyridine-functionalized silver nanoparticles. Sensors and Actuators B: Chemical, 145(1), 194-199. doi:10.1016/j.snb.2009.11.062Sakai, R., Okade, S., Barasa, E. B., Kakuchi, R., Ziabka, M., Umeda, S., … Kakuchi, T. (2010). Efficient Colorimetric Anion Detection Based on Positive Allosteric System of Urea-Functionalized Poly(phenylacetylene) Receptor. Macromolecules, 43(18), 7406-7411. doi:10.1021/ma1016852Sakai, R., Sakai, N., Satoh, T., Li, W., Zhang, A., & Kakuchi, T. (2011). Strict Size Specificity in Colorimetric Anion Detection Based on Poly(phenylacetylene) Receptor Bearing Second Generation Lysine Dendrons. Macromolecules, 44(11), 4249-4257. doi:10.1021/ma200710rIsaad, J., & Salaün, F. (2011). Functionalized poly (vinyl alcohol) polymer as chemodosimeter material for the colorimetric sensing of cyanide in pure water. Sensors and Actuators B: Chemical, 157(1), 26-33. doi:10.1016/j.snb.2011.03.022Isaad, J., & El Achari, A. (2011). Colorimetric sensing of cyanide anions in aqueous media based on functional surface modification of natural cellulose materials. Tetrahedron, 67(26), 4939-4947. doi:10.1016/j.tet.2011.04.061Yao, Z., Bai, H., Li, C., & Shi, G. (2010). Analyte-induced aggregation of conjugated polyelectrolytes: role of the charged moieties and its sensing application. Chemical Communications, 46(28), 5094. doi:10.1039/c002188aKrishnamurthi, J., Ono, T., Amemori, S., Komatsu, H., Shinkai, S., & Sada, K. (2011). Thiourea-tagged poly(octadecyl acrylate) gels as fluoride and acetate responsive polymer gels through selective complexation. Chem. Commun., 47(5), 1571-1573. doi:10.1039/c0cc03256eVallejos, S., Estévez, P., García, F. C., Serna, F., de la Peña, J. L., & García, J. M. (201

Micro and Nanotechnologies for Biotechnology

Countless healthcare and biomedical solutions with high impact in terms of timely diagnostics, therapeutic success, patient comfort or financial sustainability of healthcare systems rely on micro- and nanotechnologies. Thus, it is not at all exaggerate to claim that such technologies play in current days a tremendous role with respect to improving the quality of our life, health and well-being, which are the main priorities of modern science. This volume illustrates these statements, addressing highly significant scientific subjects from diverse areas of micro- and nanotechnologies for biotechnology. Authoritative voices in their fields present in this volume their work, or review recent trends, concepts and applications, in a manner that is accessible to a broad readership audience from both within and outside their specialist area

Development of a rapid screening technique for contaminants in environmental monitoring and regulation

Rapid screening technique is important and efficient for routine monitoring of chemical pollutants, risk assessment and decision making in dealing with contaminants in waters and soils. The focus of this thesis is on developing simple and rapid screening methods based on the diffusive gradients in thin films (DGT) technique to assess the concentration of phosphorus and metals qualitatively and quantitatively. Firstly, a rapid detection technique for phosphorus based on Metsorb DGT devices and a colour imaging method using the conventional molybdenum blue were developed and fully tested under different conditions. The fully quantitative interpretation of the P concentration can be assessed in the linear range of 0.1 to 1.02 μg cm-2 device that corresponds to the concentration range of 9 to 98 μg L-1 if the deployment time is 24 hours and the water temperature is 20oC. Secondly, digital colorimetric analysis using a flat-bed scanner was utilised to quantify the Cu, Ni, and Co in water following the DGT uptake of metals by Chelex resin gel without involving further reactive reagents. The fully quantitative interpretation of the Cu, Ni, and Co concentration can be assessed in the linear range of 1.5 to 165 μg cm-2 , 2.7 to 153 μg cm-2 , and 1.6 to 159.2 μg cm-2 , respectively, which correspond to the concentration range of 0.05 to 5 mg L-1 for all three metals if the deployment time is 24 hours and the water temperature is 20oC. Thirdly, a rapid screening technique for Cr(VI) using DGT and a high-resolution CID base on the surface colouration of the N-Methyl-D-glucamine (NMDG) binding gel has been developed. The relationship between the accumulation of Cr(VI) in NMDG gels and the corresponding change in grayscale intensity was well fitted using a quintic polynomial. The fully quantitative interpretation of the Cr(VI) concentration can be assessed in the linear range of 0.31 to 2.47 μg cm-2 which correspond to the concentration range of 12.5 to 150 μg L-1 if the deployment time is 24 hours and the water temperature is 20oC. This study has formulated a DGT deployment guide list to determine whether the concentration of metals has exceeded the maximum contaminant level allowed based on the regulation standards in different countries and regions. The use of both a simple visual inspection and a scanner for DGT devices at different deployment times and different temperatures will be considered for this list. Moreover, the rapid screening technique has been evaluated in water and soil from five regions in China. Furthermore, a novel approach with biological material incorporated in the DGT (Bio-DGT) was developed to measure the concentrations and toxicity of metals at the same time in water and soil. The new method immobilised a whole-cell toxicity bioreporter, ADPWH_recA, into a thin layer of agarose gel to replace the polyacrylamide gel that is commonly used in DGT. The test results indicated that the concentrations of metals measured by Bio-DGT and the cell free DGT have no significant differences during a 7-day deployment in synthetic water. A positive metal exposure relationship was shown between Bio-DGT accumulation and biological response. Bio-DGT showed a stable response to heavy metals under a wide range of pH and ionic strength. The bioluminescent signal of Bio-DGT was maintained at a high level during up to 30 days of storage. The deployment of Bio-DGT devices in field soils collected from China allowed the measurement of both the available concentration and the toxicity of metals. It indicated that the new Bio-DGT can assess the bioavailability and toxicity of metals at the same time. The newly developed rapid screening technique for P and metals were applied in waters and soils in situ in 5 different regions of China. It showed the concentrations of P in most of the monitored waters in Beijing were low and the quality of the waters has reached the Chinese water quality standards for surface water. The concentration of DGT-measured P in the two main rivers run through Tianjing were higher than the national water standard in China. The concentrations of Cu in monitored aquatic systems of all field areas have also reached the Chinese water quality standards for surface water

Reconnaissance électrochimique supramoléculaire d'anions d'intêret biologique à l'aide de récepteurs dendritiques et à base de nanoparticules d'or

Nous avons synthétisé trois types de dendrimères différents: * des dendrimères à coeur colloïdal d'or (liaisons de coordination), * des dendrimères "classiques" (liaisons covalentes), * des dendrimères supramoléculaires (liaisons hydrogène). Leur étude pour la reconnaissance d'oxoanions, principalement d'H2PO4- et de l'ATP2-, a montré quelques différences intéressantes: * Les nanoparticules d'or dendronisées peuvent être utilisées pour la préparation d'électrodes modifiées recyclables. * Les dendrimères possédant des fonctions ferrocényles perméthylées sont plus stables et permettent un titrage plus "propre" des anions. * Les dendrimères formés par liaisons hydrogène possèdent des propriétés particulières de reconnaissance au cours du titrage des anions étudiés. La facilité d'accès à des objets moléculaires avec un grand nombre de fonctions périphériques, offertes par les colloïdes d'or dendronisés et par les dendrimères supramoléculaires nous ouvre le champs à de nombreuses autres idées d'assemblages. Et la reconnaissance de l'ATP2- nous invite à tendre encore plus vers la biologie et vers des études en milieu aqueux