Nicholas J. Goddard

The resonant mirror: a novel optical biosensor for direct sensing of biomolecular interactions Part I: Principle of operation and associated instrumentation

Abstract The Resonant Mirror biosensor is a new design of optical sensor, aimed at combining the improved sensitivity of waveguide sensors with the simplicity of fabrication and ease of use associated with surface plasmon sensors. It uses the evanescent wave associated with a dielectric resonant structure to probe reactions occurring in a sensing layer, deposited within a few hundred nanometers of the device surface and is aimed at the real time monitoring of biological assays, without use of labelled molecules. This paper describes the operation of the device and performance features relevant to its use as a biosensor.

Development of a green fluorescent protein reporter for a yeast genotoxicity biosensor

A reporter system, constructed for a laboratory screen for new genes involved in DNA repair in the brewers yeast Saccharomyces cerevisiae, has been developed for use in a genotoxicity biosensor. The strain produces green fluorescent protein (yEGFP) when DNA damage has occurred. yEGFP is codon optimised for yeasts. The reporter does not respond to chemicals which delay mitosis, and responds appropriately to the genetic regulation of DNA repair. Data is presented which demonstrate strain improvements appropriate to biosensor technology: improved signal to noise ratio, ease of data collection and uncomplicated material handling.

Photochemistry of refractive index structures in poly(methyl methacrylate) by femtosecond laser irradiation

Femtosecond, subablation threshold photomodification of poly(methyl methacrylate) (PMMA) at 387 nm is explored to enable fabrication of optical components. Volatile fragment analysis (thermal desorption gas chromatography-mass spectrometry) and molecular weight distribution monitoring (size exclusion chromatography) suggest photochemical modification, involving direct cleavage of the polymer backbone and propagation via chain unzipping under formation of monomers, similar to the pyrolytic degradation of PMMA. Waveguides were produced in undoped, clinical-grade PMMA, showing an increased refractive index in the laser focal region (Dnmax=4x10(-3)).

Bidirectional isotachophoresis on a planar chip with integrated conductivity detection.

The use of a miniaturised planar separation device with integrated conductivity detection for performing bidirectional isotachophoresis (ITP) is described. The chips were produced in poly(methyl methacrylate) (PMMA) using a milling procedure. To enable bidirectional ITP the devices were designed to inject samples into the centre of the section channel and incorporated two integrated on-column conductivity detectors, positioned at opposite ends of this channel. When used with a hydrodynamic sample transport system the devices were used for the analysis of a range of small ions: NH4+; Na+; Mg2+; Ca2+; Li+; NO3-; ClO4-; SO4(2-); F-. Results sucessfully achieved included the simultaneous separation of three anions and three cations.

Miniaturised isotachophoretic analysis of inorganic arsenic speciation using a planar polymer chip with integrated conductivity detection

A new method allowing the analysis of inorganic arsenic species using isotachophoresis has been developed. This method has been shown to be suitable for use on both miniaturised planar polymer separation devices and capillary scale devices. A poly(methyl methacrylate) chip with integrated conductivity electrodes has been successfully used for the rapid analysis of inorganic arsenic species in under 600 s. Limits of detection of 1.8 mg l(-1) and 4.8 mg l(-1) for arsenic(V) and arsenic(II), respectively, have been achieved with the miniaturised device. The device has also been used to perform the simultaneous separation of arsenic(III), arsenic(V), antimony(III), molybdenum(VI) and tellurium(IV).

Real-time biomolecular interaction analysis using the resonant mirror sensor

The resonant mirror is a planar waveguide optical sensor that uses frustrated total internal reflection to couple light in and out of the waveguide layer. As a biosensor, the device exploits the highly selective binding interactions between pairs of biomolecules such as enzyme–substrate, antibody–antigen, hormone–receptor and DNA–DNA (DNA = deoxyribonucleic acid). As many of these species have no absorption bands at convenient (visible) wavelengths, refractive index sensing is employed to detect the displacement of water by the higher refractive index analyte as it interacts with its immobilized binding partner. The sensor chip construction is relatively simple, using techniques developed for the production of anti-reflection coatings and interference filters. Preliminary experimental results are presented from two instrumental configurations using a single type of sensor chip. Both types of instrument use linear or two-dimensional charge coupled device (CCD) arrays as detectors, thus avoiding the use of moving parts in the instrumentation.

Optimisation of secondary electrospray ionisation (SESI) for the trace determination of gas-phase volatile organic compounds

An electrospray ionisation triple quadrupole mass spectrometer (Varian 1200 L) was modified to accept nitrogen samples containing low concentrations of volatile organic compounds. Six candidate probe compounds, methyl decanoate, octan-3-one, 2-ethylhexanoic acid, 1,4-diaminobutane, dimethyl methylphosphonate, and 2,3-butanediol, at concentrations below 50 ppb(v) were generated with permeation tubes in a test atmosphere generator. The concept of using a set of molecular probes to evaluate gas-phase electrospray ionisation of volatile analytes was assessed and the feasibility of adopting a unified ionisation approach for gas and liquid contamination of exobiotic environments established. 450 experiments were run in a five-replicate, fifteen-level, three-factor, central-composite-design with exponential dilution for each of the six probe compounds studied. The three factors studied were ionisation voltage, drying-gas flow and nebulising-gas flow. Parametric modelling by regression analysis enabled the differences in the ionisation behaviours of the probe compounds to be described by the optimisation models. Regression coefficients were in the range 0.91 to 0.99, indicating satisfactory levels of precision in the optimisation models. A wide range in ionisation efficiency was observed, with different optimised conditions required for the probe compounds. It was evident that no one factor appeared to dominate the response and the different factors produced different effects on the responses for the different molecules. 1,4-Butanediamine and dimethyl methylphosphonate required significantly lower ionisation voltages (1.2 kV) than the other four, which achieved optimised sensitivity towards the maximum voltage used in this design (5 to 6 kV). Drying-gas flow rates were found to be more important than nebulising-gas flow rates. However, variations in the constant term B(0) in the optimisation models indicated that other factors, not included in this study, were also likely to be involved in the ionisation process. Electrolyte-flow rate and ionisation temperature were proposed for follow up studies. Exponential dilution data indicated sensitive and analytically useful responses in the target range of 5 to 50 ppb(v) for all six compounds. Significantly, responses were seen at concentrations significantly below 5 ppb(v), with sub ppt(v) responses observed for 1,4-butanediamine, 2-ethylhexanoic acid, dimethylmethylphosphonate, and 1,3-butanediol. Responses in the ppt(v) to ppb(v) range were observed for the remaining two compounds. The observations from this study demonstrated the utility of adopting a set of probe compounds to evaluate electrospray ionisation performance for volatile organic compound based assays; indicated the existence of multiple ionisation mechanisms; and revealed potential sensitivity at the parts per quadrillion level ppq(v).

Integrated moulded polymer electrodes for performing conductivity detection on isotachophoresis microdevices

The feasibility of using integrated injection moulded polymer electrodes as drive and detection electrodes for performing miniaturised isotachophoresis (ITP) separations with conductivity detection has been demonstrated. Injection moulded electrodes were produced from three different grades of carbon-filled polymer. Two of the electrode designs were found to be suitable for performing on-chip conductivity detection. The high-voltage characteristics of the microdevices were found to be suitable for performing ITP, with a power dissipation up to 1.4 W m(-1) being achieved. Three model separations are presented to demonstrate the separation capability of the miniaturised injection moulded devices. Three anionic dyes, two inorganic anions and a mixture of eight alkaline earth, transition and lanthanide metal cations were analysed.

Grating coupled leaky waveguide micro channel sensor chips for optical analysis

Abstract Injection moulded polymer leaky waveguide devices are described that enable on-chip fluorescence and absorption measurements to be undertaken on-line in micro total analysis systems. Embossed diffractive elements are incorporated such that effective in- and out-coupling of light from flow channels is achieved without the need for prism coupling and index matching solutions. The system is demonstrated using laser and LED sources to obtain fluorescence and absorption spectra in the visible region.

Precision milled flow-cells for chemiluminescence detection

Novel flow-cells with integrated confluence points and reaction channels designed for efficient mixing of fast chemiluminescence systems were constructed by machining opposing sides of a polymer chip and sealing the channels with transparent epoxy-acetate films. A hole drilled through the chip provided the conduit from the confluence point on one side to the centre of the reaction zone on the other side, allowing rapid presentation of the reacting mixture to the photodetector. The effectiveness of each flow-cell was evaluated by comparing the chemiluminescence intensity using flow injection analysis methodology, and examining the distribution of light emanating from the reaction zone (captured by photography in a dark room) when the reactants were continuously merged. Although previously reported chemiluminescence detectors constructed by machining channels into polymers have almost exclusively been prepared using transparent materials, we obtained far greater emission intensities using an opaque white chip with a thin transparent seal, which minimised the loss of light through surfaces not exposed to the photomultiplier tube. Furthermore, this approach enabled the exploration of reactor designs that could not be incorporated in traditional coiled-tubing flow-cells.