Towards single-spot multianalyte molecular beacon biosensors

The separate developments of microarray patterning of DNA oligonucleotides, and of DNA hairpins as sensitive probes for oligonucleotide identification in solution, have had a tremendous impact on basic biological research and clinical applications. Herein, we will discuss several successful efforts to develop oligonucleotide sensors based on the surface immobilization of functionalized DNA hairpins. We also will discuss the development of prototypical single-spot multianalyte “Molecular Beacon” biosensors. Importantly, we show that organic fluorophores will likely be inadequate in moving this technology forward and new approaches, such as the use of nanotechnology, will be needed.     

A resistive-pulse sensor chip for multianalyte immunoassays

MultiAnalyte immunoassays are often required to diagnose a pathologic condition. Here, we show how resistive-pulse sensing and multiple artificial pores can be integrated together on a single chip to detect different antigens rapidly and simultaneously. We use multiple pores on a single chip to detect the size change of latex colloids upon specific antigen-antibody binding on the colloid surface. As a proof-of-principle, we demonstrate our ability to detect simultaneously human G-CSF and GM-CSF antigens on a single chip. Our novel technique is a scalable technology that can lead to the sensing of at least N2 antigens simultaneously with an N N array of pores on a single chip.     

Single molecular multianalyte (Ca2+, Mg2+) fluorescent probe and applications to bioimaging

Intracellular signal transduction relies on spatial and temporal signal transmitter dynamics. To clarify the correlations of these transmitter molecules, multicolor-imaging has been widely used. However, in the case of applying multiple indicators in a cell, spectral overlap of the indicators prevents accurate quantitative analysis. Moreover, the invasive (toxic) effect, the localization, the metabolism, as well as photobleaching of these indicators complicate the situation. Here, we show that single-molecular multifluorescent probes can overcome these problems. While intracellular calcium plays a critical role as a signal transmitter and magnesium acts as a cofactor in many situations, the correlations between the two cations are now the main issue. We designed and synthesized a Ca2+-Mg2+ responsive multifluorescent probe, KCM-1. KCM-1 shows a spectral blue shift upon complexation to Ca2+ and a red shift to the presence of Mg2+. With data analyzed at different excitation wavelengths, the concentrations of Ca2+ and Mg2+ are simultaneously quantified. Furthermore, by using the AM-ester method, intracellular Ca2+ and Mg2+ concentrations are simultaneously imaged. Such a type of intracellular multiple analyte imaging by a single-molecular multifluorescent probe is successfully demonstrated for the first time.     

Organometallic and related imidotitanium compounds containing a pendant arm functionalised benzamidinate ligand

Chloride ligand substitution reactions of tert-butyl- and arylimido-titanium complexes supported by the pendant arm functionalised N-trimethylsilyl benzamidinate ligand Me₃SiNC(Ph)NCH₂CH₂CH₂NMe₂ are described. Reaction of previously-described [Ti(N^tBu){Me₃SiNC(Ph)NCH₂CH₂CH₂NMe₂}Cl] (1) with PhLi afforded thermally sensitive [Ti(N^tBu){Me₃SiNC(Ph)NCH₂CH₂CH₂NMe₂}Ph] (2). The corresponding reaction of 1 with MeLi afforded [Ti(N^tBu){Me₃SiNC(Ph)NCH₂CH₂CH₂NMe₂}Me] (3) detected by ¹H-NMR spectroscopy but this compound could not be isolated. Reaction of 1 with LiCH₂SiMe₃ gave a complex mixture, but with LiN(SiMe₃)₂ and LiO-2,6-C₆H₃Me₂ the compounds [Ti(N^tBu){Me₃SiNC(Ph)NCH₂CH₂CH₂NMe₂}X] (X=N(SiMe₃)₂ (4) or O-2,6-C₆H₃Me₂ (5)) were isolated. The X-ray structure of 5 was determined. Reaction of the homologous compound [Ti(N^tBu){Me₃SiNC(Ph)NCH₂CH₂NMe₂}Cl] (6) (containing a 2-carbon atom chain in the pendant arm) with MeLi or PhLi were unsuccessful although the aryloxide compound [Ti(N^tBu){Me₃SiNC(Ph)NCH₂CH₂NMe₂}(O-2,6-C₆H₃Me₂)] (7) could be isolated from the reaction of 6 with LiO-2,6-C₆H₃Me₂. Reaction of the 3-carbon pendant arm arylimido compound [Ti(N-2,6-C₆H₃Me₂){Me₃SiNC(Ph)NCH₂CH₂CH₂NMe₂}Cl] (8) with MeLi afforded thermally sensitive [Ti(N-2,6-C₆H₃Me₂){Me₃SiNC(Ph)NCH₂CH₂CH₂NMe₂}Me] (9), and although the analogous phenyl homologue was elusive, the aryloxide derivative [Ti(N-2,6-C₆H₃Me₂){Me₃SiNC(Ph)NCH₂CH₂CH₂NMe₂}(O-2,6-C₆H₃Me₂)] (10) was successfully isolated and structurally characterised. Comparison of the X-ray structures of 5 and 10 show unexpectedly large differences between the TiNR and TiOAr bond lengths in the two compounds.     

Diphosphine-phosphenium coordination complexes representing monocations with pendant donors and ligand tethered dications

Homoatomic P-P coordinate bonding is exploited to prepare the first examples of triphosphorus monocations and tetraphosphorus dications using dimethylphosphenium or diphenylphosphenium Lewis acceptors with diphosphinomethane, diphosphinoethane, diphosphinohexane, or diphosphinobenzene ligands. Solid-state structures and spectroscopic characterization data for complexes involving bis(diphenylphosphino)methane ligands show coordination of only one donor site of the diphosphine ligand in the monocations, and chelate complexation is not observed. Tetraphosphorus dications are observed with longer diphosphines, in which the ligand tethers two phosphenium acceptors. The structural preferences between monocations with pendant phosphines and tethered dications are dependent on intramolecular steric interactions and the flexibility of the tether.     

Development of multianalyte sensor arrays composed of chemically derivatized polymeric microspheres localized in micromachined cavities.

The development of a chip-based sensor array composed of individually addressable polystyrene-poly(ethylene glycol) and agarose microspheres has been demonstrated. The microspheres are selectively arranged in micromachined cavities localized on silicon wafers. These cavities are created with an anisotropic etch and serve as miniaturized reaction vessels and analysis chambers. A single drop of fluid provides sufficient analysis media to complete approximately 100 assays in these microetch pits. The cavities possess pyramidal pit shapes with trans-wafer openings that allows for both fluid flow through the microreactors/analysis chambers and optical access to the chemically sensitive microspheres. Identification and quantitation of analytes occurs via colorimetric and fluorescence changes to receptor and indicator molecules that are covalently attached to termination sites on the polymeric microspheres. Spectral data are extracted from the array efficiently using a charge-coupled device allowing for the near-real-time digital analysis of complex fluids. The power and utility of this new microbead array detection methodology is demonstrated here for the analysis of complex fluids containing a variety of important classes of analytes including acids, bases, metal cations, metabolic cofactors, and antibody reagents.     

A benzimidazole-based single molecular multianalyte fluorescent probe for the simultaneous analysis of Cu and Fe

We synthesized a novel receptor with benzimidazole moieties in a tripodal framework. The receptor displays rarely observed metal specific fluorescence enhancement at two different wavelengths. The receptor was investigated for the simultaneous analysis of Cu²⁺ and Fe³⁺ and successfully quantified the ions without interference over a wide concentration range.     

DommiMOE: an implementation of ligand field molecular mechanics in the molecular operating environment

The ligand field molecular mechanics (LFMM) model, which incorporates the ligand field stabilization energy (LFSE) directly into the potential energy expression of molecular mechanics (MM), has been implemented in the "chemically aware" molecular operating environment (MOE) software package. The new program, christened DommiMOE, is derived from our original in-house code that has been linked to MOE via its applications programming interface and a number of other routines written in MOE's native scientific vector language (SVL). DommiMOE automates the assignment of atom types and their associated parameters and popular force fields available in MOE such as MMFF94, AMBER, and CHARMM can be easily extended to provide a transition metal simulation capability. Some of the unique features of the LFMM are illustrated using MMFF94 and some simple [MCl)]2- and [Ni(NH3)n]2+ species. These studies also demonstrate how density functional theory calculations, especially on experimentally inaccessible systems, provide important data for designing improved LFMM parameters. DommiMOE treats Jahn-Teller distortions automatically, and can compute the relative energies of different spin states for Ni(II) complexes using a single set of LFMM parameters.     

Dimetallic complexes of a structurally versatile pyridazine-containing Schiff-base macrocyclic ligand with pendant pyridine arms

The new [2 + 2] Schiff-base macrocyclic ligand L2, containing pyridazine head units and pyridine pendant arms, was synthesised as [Ba(II)2L2(ClO4)4(OH2)] 1 from the barium(II) ion templated condensation reaction of 3,6-diformylpyridazine and N1-(2-aminoethyl)-N1-(methylene-2-pyridyl)-ethane-1,2-diamine. Subsequent transmetallation reactions of 1 with copper(II), iron(II) and manganese(II) perchlorates led to the formation of [Cu(II)2L2](ClO4)4.2MeCN 2, [Fe(II)2L2(MeCN)2](ClO4)4 3 and two manganese complexes, 4 and 5, with the same formula, [Mn(II)2L2(MeCN)(OH2)](ClO4)4, but slightly different crystal structures, respectively. Single-crystal X-ray structural analyses reveal the variety of structures which can be supported by L2 in order to meet the coordination environment preferences of the incorporated metal ions. The barium(II) ions in 1 have an irregular ten-coordinate geometry whereas the copper(II) ions in 2 have a square pyramidal geometry and the iron(II) ions in 3 have an octahedral geometry, while in 4 and 5 every manganese(II) ion is seven-coordinate and the environment can be best described as distorted pentagonal bipyramidal. In 1, 4 and 5 the pyridazine moieties bridge the metal centres [Ba(1)...Ba(2) 4.9557(3)A 1; Mn(1)...Mn(2) 4.520(1)A 4; Mn(1)[dot dot dot]Mn(2) 4.3707(8)A 5] but this is not observed in the copper(II) and iron(II) complexes, 2 and 3, in which the metal ions are well separated [Cu(1)...Cu(2) 5.9378(6)A 2; Fe(1)...Fe(2) 5.7407(12)A 3]. In the cyclic voltammogram of [Cu2(II)L2](ClO4)4.2MeCN 2 in MeCN vs. Ag/AgCl two separate reversible one-electron transfer steps are observed [E(1/2)=0.04 V, DeltaE= 0.12 V and E(1/2)= 0.20 V, DeltaE=0.12 V; K(c)=510; in this system E(1/2)(Fc+/Fc)=0.42 V and DeltaE(Fc+/Fc)=0.08 V]. The other complexes cannot be reversibly reduced/oxidised.     

Single chain magnets based on the oxalate ligand

The anionic oxalate-bridged bimetallic chain [Co(H2O)2Cr(ox)3]- shows slow relaxation of the magnetization, typical of the so-called single-chain magnets, when crystallized in segregated layers in a mixed salt with the supramolecular cations [C12H24O6K]+ and [(C12H24O6)(FC6H4NH3)]+. This is the first time that such phenomenon has been observed in an oxalate-bridge material. In view of the wide synthetic versatility exhibited by the oxalate ligand, it opens the door for the realization of a complete family of SCM materials whose physical properties might be tuned by the suitable replacement of M3+ ions within the chain. The information extracted from the systematic study of these compounds should provide important information concerning the main parameters that affect the slow-relaxation phenomena in this sort of 1D nanomagnets.     

Colorimetric sensor arrays for molecular recognition

The development of colorimetric sensor arrays for the detection of volatile organic compounds is reported. Using an array of chemo-responsive dyes, enormous discriminatory power is created in a simple device that can imaged easily with an ordinary flat-bed scanner. High sensitivities (ppb) have been demonstrated for the detection of biologically important analytes, including amines, carboxylic acids, and thiols. By the proper choice of dyes and substrate, the array can be made essentially non-responsive to changes in humidity.     

Template synthesis of a tetraazamacrocyclic ligand with two pendant pyridinyl groups: properties of the isomers of the metal-free ligand and of their first-row transition metal compounds

The one-step reaction of [Cu(en)(2)](2+) (en = 1,2-diaminoethane) with formaldehyde, ethyl 2-pyridyl acetate, and base produces a mixture of [Cu(s-pypymac)](2+) and [Cu(a-pypymac)](2+) (s-pypymac = syn-6,13-bis(2-pyridinyl)-1,4,8,11-tetraazacyclotetradecane, a-pypymac = anti-6,13-bis(2-pyridinyl)-1,4,8,11-tetraazacyclotetradecane; syn-to-anti ratio approximately 1:9) in low yield (6%). Ion exchange chromatography is used for isomer separation, and the two isomers of the metal-free ligand are obtained by reduction of the copper(II) complexes and subsequent ion exchange chromatography. Crystal structure analyses of the metal-free a-pypymac ligand, of two isomeric copper(II) compounds of a-pypymac and one of s-pypymac, and of the cobalt(III) complexes of a- and s-pypymac and nickel(II), as well as zinc(II) complexes of a-pypymac, are reported and discussed on the basis of the expectations from force field calculations and from published experimental data of the transition metal compounds of the bis-pendant amine derivative diammac.     

Copper complex of an iminodioxabicyclo[3.3.1]nonane pendant ligand: the first example of iminodioxocin bridgehead nitrogen coordination

The pentadentate N4O amine-imine-naphthol ligand (HL(D)) containing rigid 8,16-imino-8H,16H-dinaphtho[2,1-b:2,1-f][1,5]dioxocin is formed preferentially, under visible light irradiation conditions, instead of the product H3L, expected from imidazolidine ring formation. A strained chelate bite enforces the first bridgehead nitrogen coordination to a copper(II) ion from the bicyclic iminodioxocin part of the ligand as shown by X-ray crystallography.     

Cyclam (1,4,8,11-tetraazacyclotetradecane) with one methylphosphonate pendant arm: a new ligand for selective copper(ii) binding

The new ligand, [(1,4,8,11-tetraazacyclotetradecan-1-yl)methyl]phosphonic acid (H(2)te1P, H(2)L), was synthesized and its complexing properties towards selected metal ions were studied potentiometrically. The ligand forms a very stable complex with copper(ii)(logbeta(CuL)= 27.34), with a high selectivity over binding of other metal ions (i.e. logbeta(ZnL)= 21.03). The crystal structures of the free ligand (in its protonated form with bromide as counter-ion) and two copper(ii) complexes (obtained by crystallization at various pH) were determined. The free ligand adopts the common conformation for such macrocycles with the protonated nitrogen atoms in the corners of a virtual rectangle. In the trans-Br,O-[Cu(Br)(Hte1P)].H(2)O species, the central metal ion is surrounded by four in plane nitrogen atoms, one oxygen atom of the pendant moiety in the apical position and a bromide anion positioned trans to the oxygen atom, forming a distorted octahedral coordination sphere. In the compound [Cu(H(2)te1P)][Cu(Hte1P)]Br(3).6H(2)O, obtained from a highly acidic solution, the bromide anions are placed further away from the copper(ii) ion and the coordination environment (N(4)O) is thus square-pyramidal. In both structures, the protons are associated with non-coordinated phosphonate oxygen atoms.     

Sequential protonation and methylation of a hydride-osmium complex containing a cyclopentadienyl ligand with a pendant amine group

Complex OsH{eta5-C5H4(CH2)2NMe2}(P(i)Pr3)2 (1) reacts with 1 equiv of trifluoromethanesulfonic acid (HOTf) and trifluoromethanesulfonic acid-d1 (DOTf) to produce the dihydride and hydride-deuteride complexes, [OsHE{eta5-C5H4(CH2)2NMe2}(P(i)Pr3)2]OTf (E = H (2), D (2-d1), respectively. Treatment of 2 and 2-d1 with a second equivalent of HOTf gives [OsHE{eta5-C5H4(CH2)2NHMe2}(P(i)Pr3)2][OTf]2 (E = H (3), D (3-d1) as a result of the protonation of the nitrogen atom. While the hydride and deuteride ligands of 2, 2-d1, 3, and 3-d1 do not undergo any H/D exchange process with the solvent, in acetone-d6, the NH proton of 3 and 3-d1 changes places with a deuterium atom of the solvent to yield [OsHE{eta5-C5H4(CH2)2NDMe2}(P(i)Pr3)2][OTf]2 (E = H (3-Nd1), D (3-d2)). Complex 3-Nd1 can also be obtained from the treatment of complex 2 with DOTf in dichloromethane. No exchange process between the hydride and the ND positions in 3-Nd1 or between the deuteride and NH positions in 3-d1 has been observed. Treatment of 3-Nd1 and 3-d1 with sodium methoxide results in a selective reaction of the base with the ammonium group to regenerate 2 and 2-d1, respectively. Complex 1 also reacts with methyl and methyl-d3 trifluoromethanesulfonate (CH3OTf and CD3OTf, respectively) to give [OsH{eta5-C5H4(CH2)2NMe2CE3}(P(i)Pr3)2]OTf (E = H (4), D (4-d3)) as a result of the addition of the CE3 (E = H, D) group to the nitrogen atom. Complex 4 has been characterized by an X-ray diffraction analysis. It reacts with a second molecule of CH3OTf or CD3OTf to produce [OsH{eta5-C5H4(CH2)2NMe3}{CH2CH(CH3)P(i)P2}(P(i)Pr3)[OTf]2 (5). Similarly, complex 4-d3 reacts with a second molecule of CH3OTf or CD3OTf to yield [OsH{eta5-C5H4(CH2)2NMe2CD3}{CH2CH(CH3)P(i)P2}(P(i)Pr3)[OTf]2 (5-d3). In acetonitrile, complex 5 evolves to an equilibrium mixture of the acetonitrile adducts [Os{eta5-C5H4(CH2)2NMe3}(NCCH3)(P(i)Pr3)2][OTf]2 (7) and [Os{eta5-C5H4(CH2)2NMe3}(NCCH3)2(P(i)Pr3)][OTf]2 (8). In methanol or methanol-d4, complex 4 is not stable and loses trimethylamine to give the vinylcyclopentadienyl derivatives [OsHE(eta5-C5H4CH=CH2)(P(i)Pr3)2]OTf (E = H (9), D (9-d1)) as a result of the protonation or deuteration of the metallic center and a subsequent Hofmann elimination. Protonation of 4 with HOTf gives the dihydride-trimethylammonium derivative [OsH2{eta5-C5H4(CH2)2NMe3}(P(i)Pr3)2][OTf]2 (10). Treatment of 9 with sodium methoxide produces OsH(eta5-C5H4CH=CH2)(P(i)Pr3)2 (11).     

Single electron densities: a new tool to analyze molecular wavefunctions

A new partitioning scheme for the electron density of a many-electron wavefunction into single electron densities is proposed. These densities are based on the most probable arrangement of the electrons in an atom or molecule. Therefore, they contain information about the electron-electron interaction and, most notably, the Fermi hole due to the antisymmetry of the many-electron wavefunction. The single electron densities overlap and can be combined to electron pair distributions close to the qualitative electron pairs that represent, for instance, the basis of the valence shell electron pair repulsion model. Single electron analyses are presented for the water, ethane, and ethene molecules. The effect of electron correlation on the single electron and pair densities is investigated for the water molecule.     

A ratiometric fluorescent sensor for phosphates: Zn2+-enhanced ICT and ligand competition

A pyrene-terpyridine-Zn conjugate has been synthesized and characterized, where Zn2+ acts as an electron acceptor to enhance molecular ICT with a large emission red-shift (>100 nm). It showed a ratiometric fluorescence change upon addition of phosphate anions in buffered aqueous solution. The selective response to phosphates or pyrophosphates involved ICT and ligand competition processes.     

The role of pendant bases in molecular catalysts for H2 oxidation and production

The paper presents a review of recent studies of complexes of Ni(II) and Fe(II) coordinated by diphosphine ligands with amine bases incorporated into the ligand chelate rings. The role of the bases in the second coordination sphere in mediating rapid intramolecular M–H/N–H exchange as well as intermolecular exchange with protons and in promoting the coupling of proton- and electron-transfer processes has been studied. Factors that favor efficient proton relay properties for the pendant amines have been established and the information has been used to develop efficient electrocatalysts for both hydrogen oxidation and production.     

Inclusion of the ligand field contribution in a polarizable molecular mechanics: SIBFA-LF

To account for the distortion of the coordination sphere that takes place in complexes containing open-shell metal cations such as Cu(II), we implemented, in sum of interactions between fragments ab initio computed (SIBFA) molecular mechanics, an additional contribution to take into account the ligand field splitting of the metal d orbitals. This term, based on the angular overlap model, has been parameterized for Cu(II) coordinated to oxygen and nitrogen ligands. The comparison of the results obtained from density functional theory computations on the one hand and SIBFA or SIBFA-LF on the other shows that SIBFA-LF gives geometric arrangements similar to those obtained from quantum mechanical computations. Moreover, the geometric improvement takes place without downgrading the energetic agreement obtained from SIBFA. The systems considered are Cu(II) interacting with six water molecules, four ammonia or four imidazoles, and four water plus two formate anions.