Pavel Adam

Functionalized ultra-low fouling carboxy- and hydroxy-functional surface platforms: functionalization capacity, biorecognition capability and resistance to fouling from undiluted biological media.

The non-specific binding of non-target species to functionalized surfaces of biosensors continues to be challenge for biosensing in real-world media. Three different low-fouling and functionalizable surface platforms were employed to study the effect of functionalization on fouling resistance from several types of undiluted media including blood plasma and food media. The surface platforms investigated in this work included two polymer brushes: hydroxy-functional poly(2-hydroxyethyl methacrylate) (pHEMA) and carboxy-functional poly(carboxybetaine acrylamide) (pCBAA), and a standard OEG-based carboxy-functional alkanethiolate self-assembled monolayer (AT-SAM). The wet and dry polymer brushes were analyzed by AFM, ellipsometry, FT-IRRAS, and surface plasmon resonance (SPR). The surfaces were functionalized by the covalent attachment of antibodies, streptavidin, and oligonucleotides and the binding and biorecognition characteristics of the coatings were compared. We found that functionalization did not substantially affect the ultra-low fouling properties of pCBAA (plasma fouling of ~20 ng/cm(2)), a finding in contrast with pHEMA that completely lost its resistance to fouling after the activation of hydroxyl groups. Blocking a functionalized AT-SAM covalently with BSA decreased fouling down to the level comparable to unblocked pCBAA. However, the biorecognition capability of blocked functionalized AT-SAM was poor in comparison with functionalized pCBAA. Limits of detection of Escherichia coli O157:H7 in undiluted milk were determined to be 6×10(4), 8×10(5), and 6×10(5) cells/ml for pCBAA, pHEMA, and AT-SAM-blocked, respectively. Effect of analyte size on biorecognition activity of functionalized coatings was investigated and it was shown that the best performance in terms of overall fouling resistance and biorecognition capability is provided by pCBAA.

Compact surface plasmon-enhanced fluorescence biochip

A new concept of compact biochip for surface plasmon-enhanced fluorescence assays is reported. It takes advantage of the amplification of fluorescence signal through the coupling of fluorophore labels with confined and strongly enhanced field intensity of surface plasmons. In order to efficiently excite and collect the emitted fluorescence light via surface plasmons on a metallic sensor surface, (reverse) Kretschmann configuration is combined with diffractive optical elements embedded on the chip surface. These include a concentric relief grating for the imaging of highly directional surface plasmon-coupled emission to a detector. Additional linear grating is used for the generating of surface plasmons at the excitation wavelength on the sensor surface in order to increase the fluorescence excitation rate. The reported approach offers the increased intensity of fluorescence signal, reduced background, and compatibility with nanoimprint lithography for cost-effective preparation of sensor chip. The presented approach was implemented for biosensing in a model immunoassay experiment in which the limit of detection of 11 pM was achieved.

Surface plasmon-coupled emission on plasmonic Bragg gratings

Surface plasmon-coupled emission (SPCE) from emitters in a close proximity to a plasmonic Bragg grating is investigated. In this study, the directional fluorescence emission mediated by Bragg-scattered surface plasmons and surface plasmons diffraction cross-coupled through a thin metallic film is observed by using the reverse Kretschmann configuration. We show that controlling of dispersion relation of these surface plasmon modes by tuning the refractive index at upper and lower interfaces of a dense sub-wavelength metallic grating enables selective reducing or increasing the intensity of the light emitted to certain directions. These observations may provide important leads for design of advanced plasmonic structures in applications areas of plasmon-enhanced fluorescence spectroscopy and nanoscale optical sources.

Surface plasmon resonance optical cavity enhanced refractive index sensing

We report on a method for surface plasmon resonance (SPR) refractive index sensing based on direct time-domain measurements. An optical resonator is built around an SPR sensor, and its photon lifetime is measured as a function of loss induced by refractive index variations. The method does not rely on any spectroscopic analysis or direct intensity measurement. Time-domain measurements are practically immune to light intensity fluctuations and thus lead to high resolution. A proof of concept experiment is carried out in which a sensor response to liquid samples of different refractive indices is measured. A refractive index resolution of the current system, extrapolated from the reproducibility of cavity-decay time determinations over 133 s, is found to be about 10(-5) RIU. The possibility of long-term averaging suggests that measurements with a resolution better than 10(-7) RIU/√Hz are within reach.

Spectroscopy of Bragg-scattered surface plasmons for characterization of thin biomolecular films.

We report a new approach to characterization of thin (bio)molecular films based on spectroscopy of Bragg-scattered surface plasmons (BSSPs) generated by diffraction-coupling of counterpropagating surface plasmons on a metal-coated diffraction grating. The BSSPs exhibit fields with different penetration depths into the medium adjacent to the metal and therefore exhibit unequal sensitivities to the presence of (bio)molecular films on the surface of the metal. Therefore, spectroscopy of BSSPs enables in situ observation of the formation of biomolecular films and determination of both their refractive index and thickness. We demonstrate this capacity of spectroscopy of BSSPs in a model experiment in which growth of protein layers on a gold surface is studied.

Biosensing enhancement using passive mixing structures for microarray-based sensors.

The combination of microarray technologies with microfluidic sample delivery and real-time detection methods has the capability to simultaneously monitor 10-1000 s of biomolecular interactions in a single experiment. Despite the benefits that microfluidic systems provide, they typically operate in the laminar flow regime under mass transfer limitations, where large analyte depletion layers act as a resistance to analyte capture. By locally stirring the fluid and delivering fresh analyte to the capture spot, the use of passive mixing structures in a microarray environment can reduce the negative effects of these depletion layers and enhance the sensor performance. Despite their large potential, little attention has been given to the integration of these mixing structures in microarray sensing environments. In this study, we use passive mixing structures to enhance the mass transfer of analyte to a capture spot within a microfluidic flow cell. Using numerical methods, different structure shapes and heights were evaluated as means to increase local fluid velocities, and in turn, rates of mass transfer to a capture spot. These results were verified experimentally via the real-time detection of 20-mer ssDNA for an array of microspots. Both numerical and experimental results showed that a passive mixing structure situated directly over the capture spot can significantly enhance the binding rate of analyte to the sensing surface. Moreover, we show that these structures can be used to enhance mass transfer in experiments regarding an array of capture spots. The results of this study can be applied to any experimental system using microfluidic sample delivery methods for microarray detection techniques.

Biosensor Enhancement Using Grooved Micromixers: Part II, Experimental Studies.

In this study we examine the experimental use of the staggered herringbone mixer (SHM) for the signal enhancement of a microfluidic surface plasmon resonance imaging (SPRi) affinity-based biosensor. We define the signal enhancement (Emix) as the ratio of the time-dependent slope of the sensor response of a SHM-based microfluidic channel and that of an unmixed channel; Emix is directly proportional to changes in the sensor sensitivity and inversely proportional to changes in the sensor limit of detection (LOD). Measurements were carried out for three SHM designs under a wide range of volumetric flow rates for two analytes: high diffusivity ssDNA and low diffusivity Escherichia coli bacteria. The experimental data collected in this study was found to exhibit a good match to that predicted by the numerical methods discussed in part I of this study. We found that Emix is dependent on the SHM groove geometry, the Péclet number Pe, and the overall microchannel length L; these dependencies are discussed in detail. For realistic experimental conditions, the enhancement that the SHM can provide is in the range of 1 < Emix < 5 (0% < improvement < 400%).

SPR sensor based on a bi-diffractive grating

We report a surface plasmon resonance (SPR) sensor based on two-plasmon-spectroscopy on a special bi-diffractive grating and investigate its ability to distinguish contributions to sensor response due to surface refractive index changes (i.e. binding) and due to refractive index changes in the whole sample. Theoretical analysis yielding an estimate of an error of such decomposition is presented and used to compare performance of this sensor to that of an alternative approach based on simultaneous excitation of short-range and long-range surface plasmons on a thin metallic layer.

Optical cavity-enhanced surface plasmon resonance refractive index sensing

A novel type of high-sensitivity Surface Plasmon Resonance (SPR)-based refractive index sensor is demonstrated, with a Kretschmann prism-coupling setup enclosed in an optical cavity and interrogated by a telecom wavelength laser source. Two thin layers of Au and SiO2 are deposited on the prism (SPR chip). Analogously to the sensors commonly referred as “Intensity modulated SPR sensors”, in the presented setup refractive index variations of the sample induce a shift of the SPR central wavelength, which in turn leads to a variation of the SPR chip reflectivity. Since the SPR chip is one of the cavity mirrors, the corresponding loss can be sensitively determined by measuring the internal photon lifetime by a cavity ring-down technique [1]. The interrogating laser is periodically shut down and the subsequent exponential decay of the on-resonance transmitted intensity is recorded to retrieve the ring-down time. In order to improve the repetability of the measurement and allow for long-term averaging, the laser is frequency locked to a cavity resonance by means of the Pound-Drever-Hall scheme[2]. The setup is sketched in fig.1.