Evanescent Wave Cavity Ring-down Spectroscopy Research Articles

Investigating the influence of solvent type and pH on protein adsorption onto silica surface by evanescent-wave cavity ring-down spectroscopy.

Several studies have explored the adsorption of various proteins onto solid-liquid interfaces, revealing the crucial role of buffer solutions in biological processes. However, a comprehensive evaluation of the buffer's influence on protein absorption onto fused silica is still lacking. This study employs evanescent-wave cavity ring-down spectroscopy (EW-CRDS) to assess the influence of buffer solutions and pH on the adsorption kinetics of three globular proteins: hemoglobin (Hb), myoglobin (Mb), and cytochrome c (Cyt-C) onto fused silica. The EW-CRDS tool, with a ring-down time of and a minimum detectable absorbance of , enabled precise optical measurements at solid-liquid interfaces. The three heme proteins' adsorption behavior was investigated at pH 7 in three different solvents: deionized (DI) water, tris(hydroxymethyl)-aminomethane hydrochloride (Tris-HCl), and phosphate buffered saline (PBS). For each protein, the surface coverage, the adsorption and desorption constants, and the surface equilibrium constant were optically measured by our EW-CRDS tool. Depending on the nature of each solvent, the proteins showed a completely different adsorption trend on the silica surface. The adsorption of Mb on the silica surface was depressed in the presence of both Tris-HCl and PBS buffers compared with unbuffered (DI water) solutions. In contrast, Cyt-C adsorption appears to be relatively unaffected by the choice of buffer, as it involves strong electrostatic interactions with the surface. Notably, Hb exhibits an opposite trend, with enhanced protein adsorption in the presence of Tris-HCl and PBS buffer. The pH investigations demonstrated that the electrostatic interactions between the proteins and the surface had a major influence on protein adsorption on the silica surface, with adsorption being greatest when the pH values were around the protein's isoelectric point. This study demonstrated the ability of the highly sensitive EW-CRDS tool to study the adsorption events of the evanescent-field-confined protein species in real-time at low surface coverages with fast resolution, making it a valuable tool for studying biomolecule kinetics at solid-liquid interfaces.

Evanescent wave cavity ring-down spectroscopy based interfacial sensing of prostate-specific antigen

Evanescent wave cavity ring-down spectroscopy (EW-CRDS) is implemented as a sensitive probe for the detection of prostate-specific antigen (PSA) with a sub-femtomolar limit of detection (LOD). PSA, a glycoprotein, is an essential biomarker for scrutinizing prostate cancer. Gold nanoparticles (Au NPs) were synthesized to serve as a sensitive reporter which are strongly bound to the thiol-modified target DNA. While Au NPs/target DNA flowed via a microfluidic channel, the target DNA may hybridize with aptamer DNA immobilized on the silica surface. Thus, the adsorption kinetics of hybridization between the Au NPs/target and aptamer was measured with interfacial optical absorbance using Langmuir fit of adsorption equilibrium. The LOD of Au NPs/PSA sensor reaches 0.54 fM signifying tremendous detecting proficiency of EW-CRDS. The evanescent wave is highly sensitive for small changes in absorption of material leading to a significant variation of the ring-down time constant. Moreover, Au NPs possess inherent excellent surface plasmonic properties that made them a promising probe material for the interfacial sensing process. To assess the significance of probe material, 5-carboxytetramethylrhodamine was substituted as a reporter to determine sensing kinetics, showing LOD of 99.1 fM. EW-CRDS approach could be used as an efficient sensing technique for the examination of malignant diseases and many biological reactions.

Atomic hydrogen induced defect kinetics in amorphous silicon

Near-infrared evanescent-wave cavity ring-down spectroscopy (CRDS) has been applied to study the defect evolution in an amorphous silicon (a-Si:H) thin film subjected to a directed beam of atomic H with a flux of (0.4–2) × 1014 cm−2 s−1. To this end, a 42 ± 2 nm a-Si:H film was grown on the total internal reflection surface of a folded miniature optical resonator by hot-wire chemical vapor deposition. A fully reversible defect creation process is observed, with a nonlinear dependence on H flux, with a time resolution of 33 ms and a relative sensitivity of 10−7. Using polarizing optics, the CRDS signal was split into s- and p-polarized components, which, combined with E-field calculations, provides depth sensitivity. Extensive kinetic modeling of the observed process is used to determine rate constants for the hydrogen–material interactions and defect formation in a-Si:H, as well as revealing a high diffusion coefficient for atomic H on the order of 10−11 cm2 s−1. A novel reaction pathway is proposed, whereby H inserted into weak Si–Si bonds recombines with mobile H, resulting in a limited penetration depth for atomic H from the gas-phase on the order of 10–15 nm.

The evanescent-wave cavity ring-down spectroscopy technique applied to the investigation of thermally grown oxides on Si(100)

We report about the contribution of thermally grown SiOx overlayer on the SiOx/Si interface (with oxidation states Sin+, where n = 1, 2, 3, 4) to the optical losses of a resonant spectroscopic cavity. The experiments on Si oxide thin films were performed in evanescent wave for Si samples in contact with a total internal reflection surface of a BK7 prism. The evanescent field can be exploited to investigate properties and processes such as the absorption of thin film or solid/air interface reactions. The results show that the oxide overlayer thickness grows with the thermal exposure time and is limited after more than 7 h of treatment. Transmission electron microscopy has been used for the native oxide thickness measurement and angle-resolved X-ray photoelectron spectroscopy used to determine the thermal oxide thickness. A change of absorption coefficient Δα in the range 100–200 cm−1 is obtained by evanescent-wave cavity ring-down spectroscopy (EW-CRDS) for thermal silicon oxide overlayer, in agreement with the general trend from literature. The evaluation from the EW-CRDS experiments presents the used setup as a competitive method for measuring the absorption properties of thin overlayer.

DNA interaction probed by evanescent wave cavity ring-down absorption spectroscopy via functionalized gold nanoparticles

Evanescent wave cavity ring-down absorption spectroscopy (EW-CRDS) is employed to study interaction and binding kinetics of DNA strands by using gold nanoparticles (Au NPs) as sensitive reporters. These Au NPs are connected to target DNA of study that hybridizes with the complementary DNA fixed on the silica surface. By the absorbance of Au NPs, the interaction between two DNA strands may be examined to yield an adsorption equilibrium constant of 2.2×1010M−1 using Langmuir fit. The binding efficiency that is affected by ion concentration, buffer pH and temperature is also examined. This approach is then applied to the label-free detection of the DNA mutation diseases using the sandwich hybridization assay. For monitoring a gene associated with sickle-cell anemia, the detection limit and the adsorption equilibrium constant is determined to be 1.2pM and (3.7±0.8)×1010M−1, distinct difference from the perfectly matched DNA sequence that yields the corresponding 0.5pM and (1.1±0.2)×1011M−1. The EW-CRDS method appears to have great potential for the investigation of the kinetics of a wide range of biological reactions.

PH-dependent electronic surface spectra of chromophore species in the charged silica–water interface

The structure of the pH-dependent silica–water interface has been probed with two charged counter-ion chromophores Crystal Violet (CV) and Malachite Green (MG) by evanescent wave cavity ring-down electronic spectroscopy. The electronic spectra of these species in the charged interface have been measured directly over the wavelength range 570–680 nm. The effective CV interfacial counter-ion concentration is 185-fold larger than the bulk with a fully charged silica surface at pH 9, corresponding to a surface concentration of 0.26 molecules nm−2. The chromophore–silica surface association and dissociation kinetics indicate a binding energy at pH 9 >28 kJ mol−1 for each chromophore: a loosely bound molecular layer. The electronic spectrum of CV in the interface shows two maxima: one feature is blue-shifted 2 nm from the solution-phase maximum at 583 nm, and the second feature is red-shifted 24 nm. A Lorentzian deconstruction of the spectra indicates that they are consistent with two molecular environments: one closely associated with the surface sharing delocalised electrons and a second confined to the interface but randomly oriented.

Evanescent-wave cavity ring-down spectroscopy applied to electrochemical ion transfer at liquid–liquid interfaces

Cavity Ring-Down Spectroscopy has been successfully applied to investigate liquid–liquid interfaces in an electrochemical environment. It has been demonstrated that this technique is very sensitive to detect changes in the concentration of species at the interface which absorb at the incident wavelength. Thus, we have shown that this technique can be employed to follow diverse processes at liquid–liquid interfaces involving concentration changes produced by the applied potential. In our contribution, we show for the first time a spectroscopic response of Evanescent Wave Ring-Down Spectroscopy directly originated by charging/discharging processes and ion transfer at the electrochemical double layer of a liquid–liquid interface.

Interaction between crystal violet and anionic surfactants at silica/water interface using evanescent wave-cavity ring-down absorption spectroscopy

Evanescent wave-cavity ring-down spectroscopy (EW-CRDS) is employed to characterize micellization of anionic surfactants and the related capability of removing cationic substance off the silica surface. Crystal violet (CV+) cationic dye is used as a molecular probe to effectively determine critical hemimicelle concentration (HMC) of surfactants on the surface. The HMC results are 1×10−2, 4×10−3, 8×10−4, and 2.5×10−4mol/L for sodium sulfate salts with a carbon-chain length of C-10, C-12, C-14, and C-16, respectively. A stronger hydrophobic interaction results in a less concentration required to undergo micellization. The HMC values on the surface are about half of those in solution. When NaCl solution is added, the electrolyte helps reduce the electrostatic repulsion between the anionic sulfate heads to facilitate the surfactant aggregation, and thus, the subsequent HMC is reduced. Furthermore, the probable phase change for dye-surfactant interactions on the surface at the concentration below HMC is observed, and the desorption rates of CV+ are measured as a function of concentration and carbon-chain length of surfactants above HMC. Given each surfactant concentration at its respective HMC, the corresponding desorption rates are along the order of C-12<C-14<C-16<-C-10. The trend may be realized by two competing factors of hemimicelle size and number density. The consequences help with understanding how to apply surfactant in the chromatographic separation.

Evanescent Wave Cavity Ring-Down Spectroscopy (EW-CRDS) as a Probe of Macromolecule Adsorption Kinetics at Functionalized Interfaces

Evanescent wave cavity ring-down spectroscopy (EW-CRDS) has been employed to study the interfacial adsorption kinetics of coumarin-tagged macromolecules onto a range of functionalized planar surfaces. Such studies are valuable in designing polymers for complex systems where the degree of interaction between the polymer and surface needs to be tailored. Three tagged synthetic polymers with different functionalities are examined: poly(acrylic acid) (PAA), poly(3-sulfopropyl methacrylate, potassium salt) (PSPMA), and a mannose-modified glycopolymer. Adsorption transients at the silica/water interface are found to be characteristic for each polymer, and kinetics are deduced from the initial rates. The chemistry of the adsorption interfaces has been varied by, first, manipulation of silica surface chemistry via the bulk pH, followed by surfaces modified by poly(L-glutamic acid) (PGA) and cellulose, giving five chemically different surfaces. Complementary atomic force microscopy (AFM) imaging has been used for additional surface characterization of adsorbed layers and functionalized interfaces to allow adsorption rates to be interpreted more fully. Adsorption rates for PSPMA and the glycopolymer are seen to be highly surface sensitive, with significantly higher rates on cellulose-modified surfaces, whereas PAA shows a much smaller rate dependence on the nature of the adsorption surface.

Evanescent Wave Cavity Ringdown Spectroscopy: A Platform for the Study of Supported Lipid Bilayers

Evanescent wave cavity ringdown spectroscopy (EW-CRDS) is advocated as an approach for monitoring the formation of supported lipid bilayers (SLBs) on quartz substrates in situ and for the quantitative study of fast molecular adsorption kinetics at the resulting modified biomimetic surface. This approach is illustrated using SLBs of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). Complementary atomic force microscopy (AFM) and quartz crystal microbalance with dissipation (QCM-D) measurements confirm the formation of bilayers on quartz. The subsequent interaction of the porphyrin, 5,10,15,20-tetraphenyl-21H,23H-porphine-p,p',p'',p'''-tetrasulfonic acid tetrasodium hydrate (TPPS) with the cationic bilayer-modified silica surface has been studied using EW-CRDS combined with an impinging-jet to deliver analyte to the surface in a well-defined manner. The adsorption of TPPS to the bilayer was kinetically controlled and the adsorption rate constant was found to be 1.7 (±0.6) × 10(-4) cm s(-1) from finite element modeling of the jet hydrodynamics and associated convective-diffusion equation, coupled to a first-order surface process describing adsorption. These proof-of-concept studies provide a platform for the investigation of molecular processes at biomembranes using EW-CRDS for chemical species showing optical absorbance in the visible and ultraviolet range.

Evanescent wave cavity-based spectroscopic techniques as probes of interfacial processes

Evanescent wave cavity ring-down spectroscopy (EW-CRDS) is a surface sensitive technique, which allows optical absorption measurements at interfaces with good time resolution. In EW-CRDS, a pulsed or modulated laser beam is coupled into an optical cavity which consists of at least one optical element, such as a silica prism, at the surface of which the beam undergoes total internal reflection (TIR). At the position of TIR, an evanescent field is established whose amplitude decays exponentially with distance from the boundary. This evanescent field can be exploited to investigate interfacial properties and processes such as adsorption and surface reactions, with most applications hitherto focusing on solid/liquid and solid/air interfaces. As highlighted herein, EW-CRDS is particularly powerful for investigations of interfacial processes when combined with other techniques such as basic electrochemical measurements and microfluidic or hydrodynamic techniques. In this tutorial review, the basic elements of EW-CRDS will be introduced and the relative merits of different configurations for EW-CRDS discussed, along with various aspects of instrumentation and design. The type of information which may be obtained using EW-CRDS is illustrated with a focus on recent examples such as molecular adsorption/desorption, deposition/dissolution of nanostructures and interfacial redox reactions. The comparatively new, but complementary, cavity technique of EW-broadband cavity enhanced absorption spectroscopy (EW-BB-CEAS) is also introduced and its advantages compared with EW-CRDS are discussed. Finally, future developments and trends in EW-cavity based spectroscopy are predicted. Notably, the potential for extending the technique to probe other interfaces is exemplified with a discussion of initial interfacial absorbance measurements at a water-air interface.

Probing Redox Reactions of Immobilized Cytochrome c Using Evanescent Wave Cavity Ring‐Down Spectroscopy in a Thin‐Layer Electrochemical Cell

We report the use of evanescent wave cavity ring-down spectroscopy (EW-CRDS) to monitor the reduction by ethylenediaminetetraacetic acid iron(II) complex, [FeEDTA](2-), of an adsorbed layer of oxidized cytochrome c immobilized on fused silica. The adsorption of cytochrome c at the silica-water interface was also probed using EW-CRDS and found to be in qualitative agreement with previous studies. The reduction of the adsorbed cytochrome c was achieved by using a strategically positioned electrode to electrogenerate FeEDTA(2-), which diffused to the silica surface and reduced the cytochrome c. The difference in the absorption spectra of the reduced and oxidized forms of cytochrome c at 400 nm allowed the direct monitoring of the electron transfer in real time. Using finite-element modelling, the rate constant of electron transfer (ET) between FeEDTA(2-) and cytochrome c was found to be 4.3(± 0.6) × 10(-9) cm s(-1) equivalent to 2.7(± 0.4) M(-1) s(-1). The latter value is considerably lower than previously reported ET rate constants between cytochrome c and FeEDTA(2-) in solution, which can be attributed to the confinement of the immobilized cytochrome c on the surface and possible effects from molecular crowding. This study highlights the importance of new methods which can be used to study ET at interfaces and opens up the possibility of studying ET to proteins in biologically relevant environments using EW-CRDS.

Kinetics of Porphyrin Adsorption and DNA-Assisted Desorption at the Silica−Water Interface

Evanescent wave cavity ring-down spectroscopy (EW-CRDS) has been used to study in situ the kinetics of the adsorption of 5,10,15,20-tetrakis(4-N-methylpyridiniumyl)porphyrin (TMPyP) from pH 7.4 phosphate buffer solution (PBS) to the silica-water interface and the interaction of calf thymus DNA (CT-DNA) with the resulting TMPyP-functionalized surface. TMPyP was delivered to the silica surface using an impinging jet technique to allow relatively fast surface kinetics to be accessed. Adsorption was first-order in TMPyP, and the initial adsorption rate constant at the bare surface was found to be k = (4.1 +/- 0.6) x 10(-2) cm s(-1). A deceleration in the adsorption kinetics was observed at longer times that could be described semiquantitatively using an Elovich-type kinetic expression. The limiting value of the absorbance corresponded approximately to monolayer coverage (6.2 x 10(13) molecules cm(-2)). Exposure of the TMPyP-modified silica surface to CT-DNA, achieved by flowing CT-DNA solution over the functionalized surface, resulted in efficient desorption of the TMPyP. The desorption process was driven by the interaction of TMPyP with CT-DNA, which UV-vis spectroscopy indicated involved intercalative binding. The desorption kinetics were also simulated using complementary finite element modeling of convection-diffusion coupled to a surface process.

Following interfacial kinetics in real time using broadband evanescent wave cavity-enhanced absorption spectroscopy: a comparison of light-emitting diodes and supercontinuum sources

A white light-emitting diode (LED) with emission between 420 and 700 nm and a supercontinuum (SC) source with emission between 450 and 2500 nm have been compared for use in evanescent wave broadband cavity-enhanced absorption spectroscopy (EW-BB-CEAS). The method is calibrated using a dye with known absorbance. While the LED is more economic as an excitation source, the SC source is superior both in terms of baseline noise (noise equivalent absorbances lower than 10(-5) compared to 10(-4) absorbance units (a.u.)) and accuracy of the measurement; these baseline noise levels are comparable to evanescent wave cavity ringdown spectroscopy (EW-CRDS) studies while the accessible spectral region of EW-BB-CEAS is much larger (420-750 nm in this study, compared to several tens of nanometres for EW-CRDS). The improvements afforded by the use of an SC source in combination with a high sensitivity detector are demonstrated in the broadband detection of electrogenerated Ir(IV) complexes in a thin-layer electrochemical cell arrangement. Excellent signal to noise is achieved with 10 micros signal accumulation times at a repetition rate of 600 Hz, easily fast enough to follow, in real time, solution kinetics and interfacial processes.

Kinetic and Thermodynamic Investigation of Rhodamine B Adsorption at Solid/Solvent Interfaces by Use of Evanescent-Wave Cavity Ring-Down Spectroscopy

Evanescent-wave cavity ring-down spectroscopy is applied to investigate the adsorption behavior of rhodamine B at three different interfaces. The adsorption equilibrium constant (K(ads)) and adsorption free energy of rhodamine B at the silica/methanol interface are determined to be (1.5 +/- 0.2) x 10(4) M(-1) and -23.8 +/- 0.4 kJ/mol by use of a Langmuir isotherm model. A Langmuir-based kinetic model is also developed to determine the corresponding adsorption and desorption rate constants of (1.02 +/- 0.03) x 10(2) M(-1) s(-1) and (7.1 +/- 0.2) x 10(-3) s(-1), from which K(ads) is obtained to be (1.45 +/- 0.09) x 10(4) M(-1), in agreement with the value determined under equilibrium conditions. Similarly, when rhodamine B is at the chlorotrimethylsilane-immobilized silica/methanol interface, the adsorption and desorption rate constants are determined to be (1.7 +/- 0.2) x 10(2) M(-1) s(-1) and (5.0 +/- 1.0) x 10(-3) s(-1). The subsequent K(ads) is (3.6 +/- 0.4) x 10(4) M(-1), which is larger than that at the silica/methanol interface. The former adsorption is dominated by hydrophobic interaction, while the latter is subject to electrostatic attraction. When rhodamine B is at the silica/water interface, there exist three chemical forms, including zwitterion (R(+)B(-)), cation (RBH(+)), and lactone (RBL). A combination of double-layer and Langmuir competitive models is used to fit the adsorption isotherm as a function of solution pH, yielding K(ads) of (2.5 +/- 0.2) x 10(4) M(-1) and (1.1 +/- 0.2) x 10(5) M(-1) for R(+)B(-) and RBH(+), respectively. RBL is considered to have the same K(ads) value as R(+)B(-).

Effectiveness of Charged Noncovalent Polymer Coatings against Protein Adsorption to Silica Surfaces Studied by Evanescent-Wave Cavity Ring-Down Spectroscopy and Capillary Electrophoresis

Protein adsorption to silica surfaces is a notorious problem in analytical separations. Evanescent-wave cavity ring-down spectroscopy (EW-CRDS) and capillary electrophoresis (CE) were employed to investigate the capability of positively charged polymer coatings to minimize the adsorption of basic proteins. Adsorption of cytochrome c (cyt c) to silica coated with a single layer of polybrene (PB), or a triple layer of PB, dextran sulfate (DS), and PB, was studied and compared to bare silica. Direct analysis of silica surfaces by EW-CRDS revealed that both coatings effectively reduce irreversible protein adsorption. Significant adsorption was observed only for protein concentrations above 400 microM, whereas the PB-DS-PB coating was shown to be most effective and stable. CE analyses of cyt c were performed with and without the respective coatings applied to the fused-silica capillary wall. Monitoring of the electroosmotic flow and protein peak areas indicated a strong reduction of irreversible protein adsorption by the positively charged coatings. Determination of the electrophoretic mobility and peak width of cyt c revealed reversible protein adsorption to the PB coating. It is concluded that the combination of results from EW-CRDS and CE provides highly useful information on the adsorptive characteristics of bare and coated silica surfaces toward basic proteins.

Real-Time Monitoring of Polyaniline Nanoparticle Formation on Surfaces

The formation of polyaniline (PAni) nanoparticles on silica surfaces has been monitored in situ using evanescent wave cavity ring-down spectroscopy (EW-CRDS). Aniline hydrochloride in aqueous solution at different concentrations was oxidatively polymerized using sodium persulfate. The process was found to involve the nucleation and growth of polymer nanoparticles, whose size and coverages were measured using tapping mode atomic force microscopy (TM-AFM). The formation of PAni was confirmed by replacing the silica surface with indium doped tin oxide (ITO) electrodes and subsequently running cyclic voltammetry experiments on the material deposited, which yielded the characteristic electrochemical response. The number of active groups (monomers) per polymer particle was estimated using the peak current of the cyclic voltammograms, with knowledge of the nanoparticle surface coverage from TM-AFM analysis. The quantity of material in each particle was consistent with TM-AFM height data, assuming hemispherical particle morphology. The polymerization process was found to be governed by surface-controlled kinetics, from a fixed number of particle sites of 35 (±5) μm−2. The reaction was first-order in aniline, with a rate constant for monomer addition of 0.7 (±0.4) × 10−7 cm s−1 in the limit of large particle size. For smaller nanoparticles, however, at the early stage of polymerization, the polymerization rate constant appeared to be size-dependent and to increase with decreasing nanoparticle radius (assuming a hemispherical geometry). This could also be due to a change in the morphology of the nanoparticles at the very shortest times but may indicate size-dependent polymerization kinetics.

Evanescent-wave cavity ring-down detection of cytochrome c on surface-modified prisms

Adsorption kinetics and molecular interactions on different surface interfaces are studied by means of evanescent-wave cavity ring-down spectroscopy, using total internal reflection surfaces onto which different self-assembled monolayers are covalently attached. The adsorption of cytochrome c (a positively charged, spherical heme protein) to a negatively charged bare silica surface, as well as to C 18-coated (hydrophobic) and C 3NH 2-coated (positively charged) silica have been studied. It is experimentally verified that these surface layers do not interfere with the sensitive measurement of adsorbed cyt c monolayers using the evanescent wave in a ring-down scheme. Attaching monolayers covalently to the silica total internal reflection surface is a first step towards the development of a biosensor that makes use of immobilized biomolecules for specific detection of analytes in solution.

Evanescent Wave Cavity Ring-Down Spectroscopy as a Probe of Interfacial Adsorption: Interaction of Tris(2,2′-bipyridine)ruthenium(II) with Silica Surfaces and Polyelectrolyte Films

Evanescent wave cavity ring-down spectroscopy (EW-CRDS) has been used to study the interaction of the tris(2,2'-bipyridine)ruthenium(II) complex, [Ru(bpy)(3)](2+), at both native silica surfaces and surfaces modified with polyelectrolyte films. Both poly-l-lysine (PLL) and PLL/poly-l-glutamic acid (PGA) bilayer functionalized interfaces have been studied. Concentration isotherms exhibit Langmuir-type adsorption behavior on both silica and PGA-terminated surfaces from which equilibrium constants have been derived. The pH-dependence of the [Ru(bpy)(3)](2+) adsorption to silica and the PLL/PGA film has also been investigated. For the latter substrate, the effective surface pK(a) of the acid groups was found to be 5.5. The effect of supporting electrolyte was also investigated and was shown to have a significant effect on the extent of [Ru(bpy)(3)](2+) adsorption. A thin-layer electrochemical cell arrangement, in which a working electrode was positioned just above the substrate, was used to change the solution pH in a controlled way via the potential-pulsed chronoamperometric oxidation of water. By measuring the optical absorption using EW-CRDS during such experiments, the desorption of [Ru(bpy)(3)](2+) from the surface has been monitored in real time. Experiments were carried out at different cell thicknesses and at various pulse durations. By combining data from the EW-CRDS experiments with fluorescence confocal laser scanning microscopy (CLSM) to determine the pH at the substrate surface, the pK(a) of the PLL/PGA film could be ascertained and was found to agree with the static pH isotherm measurements. These studies provide a platform for the further use of electrochemistry combined with EW-CRDS to investigate dynamic processes at interfaces.

A new approach to detecting biologically active substances with evanescent-wave cavity ring-down spectroscopy

We have shown that an amino-propyl coating, frequently used in bioanalysis to produce activated surfaces, is sufficiently transparent to allow it to be employed in evanescent-wave cavity ring-down spectroscopy (EWCRDS). Using a quartz prism with an activated surface, it was possible to detect femtomole quantities of labeled biomolecules. The target molecules were immobilized, and thus concentrated, on the total internal reflecting surface of a simple 90° prism. Initial measurements were carried out on a dye typically used for labeling biomolecules. An antigen (fibronectin) labeled with this dye, was also detected in the femtomole-range by first immobilizing the specific antibody on the activated surface.