Layne D. Williams

Biosensing based upon molecular confinement in metallic nanocavity arrays

We describe the basis for an affinity biosensor platform in which enhanced fluorescence transduction occurs through the optical excitation of molecules located within metallic nanocavities. These nanocavities are about 200 nm in diameter, are arranged in periodic or random two-dimensional arrays, and are fabricated in 70 nm thick gold films by e-beam lithography using negative e-beam resist. The experimental results show that both periodic and randomly placed metallic nanocavities can be used to enhance the fluorescence output of molecules within the cavities by about a factor of ten. In addition, the platform provides isolation from fluorescence produced by unbound species, making it suitable for real-time detection. Finally, we demonstrate the use of the platform in the real-time detection of 20-base oligonucleotides in solution.

Low noise detection of biomolecular interactions with signal-locking surface plasmon resonance.

Surface plasmon resonance (SPR) is a popular technique for label-free detection of biomolecular interactions at a surface. SPR yields quantitative kinetic association and dissociation constants of surface interactions such as the binding of two molecular species, one present in the liquid phase and the other immobilized at the surface. Current state-of-the-art SPR systems extract kinetic constants from measurements of the step response of the interaction versus time. The step response measurement is subject to the influence of noise and drift disturbances that limit its minimum-detectable mass changes. This paper presents a new SPR technique that measures the biomolecular interaction not in time but over a very narrow frequency range under periodic excitation. The measured response is, thus, locked to a very specific narrow band signal. This narrow band spectral sensing scheme has a very high degree of rejection to uncorrelated spurious signals. The signal-locked SPR technique was implemented using a chemical modulator chip connected to a set of functionalized Au sensing sites downstream. Binding experiments for a model system of carbonic anhydrase-II (CA-II) analyte and immobilized 4-(2-aminoethyl)benzenesulfonamide (ABS) ligand display a 100-fold (20 dB) improvement in the measured signal-to-noise ratio (SNR) when using the new technique compared to the SNR achieved using the conventional step response method.

Label-free detection of protein binding with multisine SPR microchips

Label-free techniques such as surface plasmon resonance (SPR) have used a step-response excitation method to characterize the binding of two biochemical entities. A major drawback of the step response technique is its high susceptibility to thermal drifts and noise which directly determine the minimum detectable binding mass. In this paper we present a new frequency-domain method based on the use of multisine chemical excitation that is much less sensitive to these disturbances. The multisine method was implemented in a PDMS microfluidic chip using a dual channel, dual multiplug chemical signal generator connected to functionalized and reference SPR binding spots. Kinetic constants for the reaction are extracted from the characteristics of the sense spot response versus frequency. The feasibility of the technique was tested using a model system of Carbonic Anhydrase-II analyte and amino-benzenesulfonamide ligand. The experimental signal to noise ratio (SNR) for the multisine measurement is about 32 dB; 7 dB higher than that observed with the single step-response method, while the overall measurement time is twice as long as the step method.

The paradox of multiplex DNA melting on a surface

Under equilibrium conditions, there are two regimes of target capture on a surface--target limited and probe limited. In the probe limited regime, the melting curve from multiplex target dissociation from the surface exhibits a single transition due to a reverse displacement mechanism of the low affinity species. The melting curve cannot be used in analytical methods to resolve heteroduplexes; only with the simplex system can proper thermodynamics be obtained.

Microarray Temperature Optimization Using Hybridization Kinetics

In any microarray hybridization experiment, there are contributions at each probe spot due to the match and numerous mismatch target species (i.e., cross-hybridizations). One goal of temperature optimization is to minimize the contribution of mismatch species; however, achieving this goal may come at the expense of obtaining equilibrium reaction conditions. We employ two-component thermodynamic and kinetic models to study the trade-offs involved in temperature optimization. These models show that the maximum selectivity is achieved at equilibrium, but that the mismatch species controls the time to equilibrium via the competitive displacement mechanism. Also, selectivity is improved at lower temperatures. However, the time to equilibrium is also extended, so that greater selectivity cannot be achieved in practice. We also employ a two-color real-time microarray reader to experimentally demonstrate these effects by independently monitoring the match and mismatch species during multiplex hybridization. The only universal criterion that can be employed is to optimize temperature based upon attaining equilibrium reaction conditions. This temperature varies from one probe to another, but can be determined empirically using standard microarray experimentation methods.

Resonant cavity optical biosensors for the detection of nucleic acid hybridization

Optical microcavities can be used to enhance the detection sensitivity of evanescent-wave fluorescence biosensors to the binding of a labeled analyte to a biospecific monolayer. The enhancement results form the buildup of intensity within the microcavity on resonance, which thereby increases fluorescence output from species specifically bound on the surface of the microcavity. Target studies are directed at nucleic acid hybridization, and initial results using high-Q dielectric microspheres have been obtained.

Development and characterization of a microheater array device for real-time DNA mutation detection

DNA analysis, specifically single nucleotide polymorphism (SNP) detection, is becoming increasingly important in rapid diagnostics and disease detection. Temperature is often controlled to help speed reaction rates and perform melting of hybridized oligonucleotides. The difference in melting temperatures, Tm, between wild-type and SNP sequences, respectively, to a given probe oligonucleotide, is indicative of the specificity of the reaction. We have characterized Tms in solution and on a solid substrate of three sequences from known mutations associated with Cystic Fibrosis. Taking advantage of Tm differences, a microheater array device was designed to enable individual temperature control of up to 18 specific hybridization events. The device was fabricated at Sandia National Laboratories using surface micromachining techniques. The microheaters have been characterized using an IR camera at Sandia and show individual temperature control with minimal thermal cross talk. Development of the device as a real-time DNA detection platform, including surface chemistry and associated microfluidics, is described.

Toward a disposable real-time DNA biosensing platform

With the goal of a portable diagnostic system in mind, we have designed a disposable platform for DNA detection. Surface micromachining using the SwIFT process at Sandia National Laboratories was used to make the new device, combining a waveguide, grating optics, heating structures, on-chip pumping, and microfluidics in a disposable package. PDMS microfluidic channels are integrated with the surface micromachined device to enable higher flow rates and added fluid complexity. Work on DNA hybridization under flow is presented, as applies to the function of the sensor. A description of the platform covering heating of the waveguide surface, laser coupling into the waveguide using grating optics, attachment chemistry for the sensor surface, and sealing of the PDMS microfluidic system to the device is given.

Real-time DNA biosensor using passive microfluidic structures

An evanescent field waveguide DNA biosensor coupled with a microfluidic sample delivery system is the platform for our studies. By employing microfluidics our aim is to reduce sample size and scale to large arrays, thereby increasing the efficiency of the sensor. We are studying the hybridization rate in small sensing zones under flow conditions, with a capture oligonucleotide immobilized on the surface of the waveguide. We are also investigating what effect passive mixing structures placed in the sensing zone have on the hybridization kinetics of the system.

Detection of Biomolecular Binding by Fourier-Transform SPR

Surface plasmon resonance (SPR) is a widely used label-free detection technique that has many applications in drug discovery, pharmacokinetics, systems biology and food science. The SPR technique measures the dynamics of a biomolecular interaction at a surface, yielding kinetic association and dissociation constants. Present SPR systems measure the step response of the interaction in time domain hence are subject to time-varying noise disturbances and drifts that limit the minimum-detectable mass changes. This paper presents a new synchronous SPR technique that measures the biomolecular interaction not in time domain, but in frequency domain with a high degree of rejection to uncorrelated spurious signals. The new technique was implemented using a PDMS microfluidic chemical signal modulator chip connected to a set of on-chip functionalized Au SPR sensing sites. Preliminary experimental spectral data for a model system of carbonic anhydrase binding demonstrates the feasibility of the new spectral technique.