3D microelectrode arrays, pushing the bounds of sensitivity toward a generic platform for point-of-care diagnostics.

The increased sensitivity of microelectrode arrays (MEAs) over macroelectrodes for biosensing is well established, and results from reducing the diffusion gradient of the target species to and from the electrode surfaces. The current study describes the fabrication and characterisation of a polymer-based MEA, which exploits the advantages of three dimensionality (3D). Firstly, the unique 3D formfactor promotes release of the gold tips from an inert layer in a controlled fashion, to form a highly reproducible array of microelectrodes in a single step. The 3D topography of the fabricated MEAs significantly enhances the diffusion profile of the target species to the electrode which results in higher sensitivity. Furthermore, the "sharpness" of the 3D structure induces differential current distribution that is focused at the apices of the individual electrodes, reducing the active area, and thereby overcoming the requirement for the electrodes to be sub-micron in size before true MEA behaviour can be achieved. The electrochemical characteristics of the 3D MEAs shows ideal micro-electrode behaviour, with a level of sensitivity of three orders of magnitude greater than that of enzyme-linked immunosorbent assays (ELISA), as the optical based gold standard. The application of the 3D MEAs uses the combination of enzyme-label and substrate approach employed in ELISAs as a generic basis for biosensing and can hence be applied to the plethora of targets that utilise the ELISA approach. As an example, the 3D MEAs are applied to the detection of RNA and demonstrate a level of detection down to single digit picomolar concentrations.

Improving nanoparticle-enhanced surface plasmon resonance detection of small molecules by reducing steric hindrance via molecular linkers.

Ultrasensitive Surface Plasmon Resonance (SPR) detection of small molecules can be achieved using nanoparticles to both enhance the SPR signals and pre-concentrate low levels of target analytes in the sample. However, the short effective penetration depth of the SPR evanescent field, and steric hindrance of binding when immobilizing small molecules on surfaces, limits the applicability of using relatively large nanoparticles (≥100 nm) for SPR detection. To overcome the issues of steric hindrance, this paper investigates the role of the molecular linkers to tether both the antibodies to the magnetic nanoparticles, and to bind the small molecules to the surface of the SPR chips. By extending the distance of the small molecule (progesterone) away from the SPR chip surface and improving the antibody orientation on the large magnetic nanoparticles, a sensitive SPR detection for progesterone was achieved in buffer (0.013 ng mL-1). The results of the SPR assay for progesterone in milk were in good correlation with ELISA results, and could be used to verify the onset of estrus in cows.

Serum metabolomics using ultra performance liquid chromatography coupled to mass spectrometry in lactating dairy cows following a single dose of sporidesmin.

INTRODUCTION: Photosensitization is a common clinical sign in cows suffering from liver damage caused by the mycotoxin sporidesmin. This disease, called facial eczema (FE), is of major importance in New Zealand. Current techniques for diagnosing animals with subclinical sporidesmin-induced liver damage (i.e. without photosensitization) are nonspecific. In addition, little is known of the mechanisms involved in sporidesmin resistance, nor the early effects seen following low-dose sporidesmin intoxication. OBJECTIVE: The objective of this study was to identify individual metabolites or metabolic profiles that could be used as serum markers for early stage FE in lactating cows. METHODS: Results are presented from a 59-day sporidesmin challenge in Friesian-cross dairy cows. Serum metabolite profiles were obtained using reversed phase ultra-performance liquid chromatography (UPLC) electrospray ionization mass spectrometry (MS) and UPLC tandem MS. Multivariate and time series analyses were used to assess the data. RESULTS: Statistical analysis, both with and without the temporal component, could distinguish the profiles of animals with clinical signs from the others, but not those affected subclinically. An increase in the concentrations of a combination of taurine- and glycine-conjugated secondary bile acids (BAs) was the most likely cause of the separation. This is the first time that MS methods have been applied to FE and that bile acids changes have been detected in cattle exposed to sporidesmin. CONCLUSIONS: It is well known that BA concentrations increase during cholestasis due to damage to bile ducts and leakage of the bile. This is the first study to investigate metabolomic changes in serum following a sporidesmin challenge. Further work to establish the significance of the elevation of individual BAs concentrations in the serum of early-stage sporidesmin-poisoned cows is necessary.

In situ surface protein conjugation of small molecules for SPR immunoassays.

Solution based protein conjugation of small molecules involves multiple steps of chemical syntheses, bio-conjugation and purifications which are labor intensive and time-consuming. Since many small molecules have limited water solubility, conjugation to a protein is also a relatively low efficiency process in aqueous solutions. In this study, a model in situ protein conjugation of small molecules was achieved onto SPR surfaces, using progesterone and ovalbumin as a model small-molecule-protein system. The in situ protein conjugation not only eliminated the requirement for wet chemistry, but also provided an easy way to optimize the assay performance and screen various molecular linkers.

Comparison of a carboxylated terthiophene surface with carboxymethylated dextran layer for surface plasmon resonance detection of progesterone.

Functionalization of a gold surface is usually accomplished by covalent binding via self-assembled monolayers (SAMs) on the gold surface, followed by attachment of flexible polymeric linker layers such as dextran hydrogels. However, these techniques require multiple steps and also have nonspecific interactions and steric problems. In this study, a self-assembled carboxylated terthiophene monolayer was formed onto a gold surface to create a sensitive and stable surface plasmon resonance (SPR) biosensing system. Compared with a commercial carboxymethyl dextran chip (CM5), the terthiophene SAM surface provided more than six times more antibody-binding signals and nearly three times the SPR assay sensitivity for progesterone (P4).

G-Quadruplex Supramolecular Assemblies in Photochemical Upconversion.

Parallel, tetramolecular G-quadruplex (G4) DNA possessing TINA monomer, (R)-1-O-[4-(1-pyrenylethynyl)phenylmethyl]glycerol, were synthesised and evaluated in complexes with tris(2,2'-bipyridine)ruthenium(II), [Ru(bpy)3 ](2+) , and the Zn(2+) derivative of 5,10,15,20-tetrakis-(1-methyl-4-pyridyl)-21 H,23H-porphine, ZnTMpyP4. UV/Vis, fluorescence, and circular dichroism (CD) spectroscopy showed that the use of G4-DNA as a template resulted in the effective communication between the ligands and the TINA molecule that was covalently attached to the 5'-end and between T and dG at the 5'-end of the dTG4 T sequence. Only one G4-DNA possessing the TINA molecule at the 5'-end of the dTG4 T sequence was able to yield a green-to-blue photochemical upconversion (PUC, λem =420 nm) in the presence of [Ru(bpy)3 ](2+) upon excitation at 500 nm. Different DNA secondary structures can thus be used in DNA-based assemblies for PUC and the way of attachment of chromophores to DNA plays a pivotal role for the creation of a photosynthetic centre.

DNA-Based Assemblies for Photochemical Upconversion.

In the present study DNA was used as a scaffold for the supramolecular assembly of organic chromophores for photochemical upconversion (PUC). Initially, a green-to-blue PUC was observed using free chromophores in solution: tris(2,2'-bipyridine)ruthenium(II), [Ru(bpy)3](2+), which acts as a long-wavelength absorber (λex = 500 nm), and an in situ energy donor to an acceptor (R)-1-O-[4-(1-pyrenylethynyl)phenylmethyl]glycerol (PEPy or TINA monomer), which acts as an annihilator and short-wavelength photoemitter (λem = 420 nm). Then, DNA duplexes possessing TINA monomers were synthesized, and complexes with [Ru(bpy)3](2+) were investigated. In contrast to the dynamic interactions of [Ru(bpy)3](2+) with TINA monomer free in solution, ground-state complex formation was the predominant mechanism of interaction between [Ru(bpy)3](2+) and DNA duplexes bearing two TINA monomers at the 5' ends as shown by fluorescence, circular dichroism (CD), and UV-vis spectroscopy studies. The use of TINA-modified DNAs led to PUC occurring at concentrations significantly lower than that for free chromophores in solution: 2.5 µM [Ru(bpy)3](2+) and 5.0 µM TINA-modified duplex in the DNA-based systems in aqueous buffer versus 46 µM [Ru(bpy)3](2+) and 4.6 mM TINA monomer for the free donor and acceptor in DCM, respectively. Providing vast capabilities of DNA in the development of novel photonic systems as a result of the controllable organization of various chromophores, this study opens a new perspective for the development of DNA-based light-harvesting systems using PUC.

In-Depth Electrochemical Investigation of Surface Attachment Chemistry via Carbodiimide Coupling.

Aminoferrocene is used as an electroactive indicator to investigate carbodiimide coupling reactions on a carboxylic acid-functionalized self-assembled monolayer. The commonly used attachment chemistry with 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) is used for surface activation. A number of conditions are investigated, including EDC and NHS concentration, buffer solutions, incubation timing, and aminoferrocene concentration. Ferrocene is a well-documented electroactive species, and the number of surface-bound ferrocene species can be calculated using electrochemical methods. This capability allows determination of optimal conditions, as well as providing a method for comparing and investigating novel carboxylated surfaces. An EDC-mediated procedure with ∼5 mM EDC and NHS (1:1) made in water, with a full acid monolayer, with 250 µM aminoferrocene for 40 min was found to give the highest ferrocene attachment. An application of this is demonstrated for preparing a probe-DNA-coated surface for DNA sensing. By backfilling with aminoferrocene, a differential quantification of the amount of probe DNA available for sensing can be obtained. This provides an elegant method to monitor an important aspect, namely, probe surface characterization, which will be highly useful for biosensing purposes.

Synthesis of ß-pyrrolic-modified porphyrins and their incorporation into DNA.

A synthetic methodology for the synthesis of various ß-pyrrolic-functionalised porphyrins and their covalent attachment to 2'-deoxyuridine and DNA is described. Palladium(0)-catalysed Sonogashira and copper(I)-catalysed Huisgen 1,3-dipolar cycloaddition reactions were used to insert porphyrins into the structure of 2'-deoxyuridine and DNA. Insertion of a porphyrin into the middle of single-stranded CT oligonucleotides possessing a 5'-terminal run of four cytosines was shown to trigger the formation of pH- and temperature-dependent i-motif structures. Porphyrin insertion also led to the aggregation of single-stranded purine-pyrimidine sequences, which could be dissociated by heating at 90 °C for 5 min. Parallel triplexes and anti-parallel duplexes were formed in the presence of the appropriate complementary strand(s). Depending on the modification, porphyrins were placed in the major and minor grooves of duplexes and were used as bulged intercalating insertions in duplexes and triplexes. In general, the thermal stabilisation of parallel triplexes possessing porphyrin-modified triplex-forming oligonucleotide (TFO) strands was observed, whereas anti-parallel duplexes were destabilised. These results are compared and discussed on the basis of the results of molecular modelling calculations.

Spectroscopic and computational study of ß-ethynylphenylene substituted zinc and free-base porphyrins.

A series of tetraphenylporphyrins appended at the ß-pyrrolic position with an ethynylphenylene- or ethynylpyridine-substituent have been subjected to spectroscopic and density functional theory (DFT) analyses. The mean absolute deviation between corresponding experimental and DFT-derived vibrational spectra is up to 10.2 cm(-1), suggesting that the DFT B3LYP/6-31G(d) method provides an accurate model of the ß-substituted porphyrin systems. The configuration interactions that give rise to prominent electronic absorptions have been calculated using time-dependant DFT (TD-DFT) and have been rationalized with reference to the energy and topology of DFT calculated molecular orbitals. As the electron withdrawing capacity of the ß-substituent increases the LUMO orbital gains appreciable amplitude over the substituent moiety and is stabilised. This represents a departure from the assumptions underpinning the Gouterman four-orbital model, resulting in atypical electronic absorption spectra. This phenomenon is also manifested in the enhancement patterns of the resonance Raman spectra insofar as B-band excitation engenders an enhancement of substituent based modes. These observations demonstrate that the ß-substituent exerts an appreciable electronic influence on the porphyrin π-electron system and provides a means of introducing charge-transfer character to prominent electronic transitions.

Sensitive determination of estriol-16-glucuronide using surface plasmon resonance sensing.

For the quantitative evaluation of low levels of an estriol metabolite of estriol (estriol-16-glucuronide (E3-16G)) in liquid media, we developed a simple and highly sensitive immunoassay using a surface plasmon resonance (SPR) biosensor which did not require any time-consuming sample pretreatment steps. E3-16G was conjugated to ovalbumin (OVA) through an oligoethylene glycol (OEG) linker to form protein conjugates (E3-16G-OEG-OVA), which were then immobilized on a carboxymethyl dextran-coated sensor chip via amine coupling to develop inhibition immunoassays. A limit of detection (LOD) of 76 pg/mL was achieved using a rabbit anti-sheep primary antibody as a binding agent. The detection limit was further improved by using synthesized gold colloids (15 nm) as high mass labels conjugated to the primary antibody. In this Au nanoparticle-enhanced assay, the concentration of E3-16G in aqueous samples could be determined in 7.5 min at a level as low as 14 pg/mL. In addition, the high stability of the E3-16G-OEG-OVA surface gave no obvious drop in antibody-binding capability after more than 1000 binding/regeneration cycles which significantly lowered the research cost.