Backscattering Interferometry Research Articles

The potential of backscattering interferometry as an in vitro clinical diagnostic tool for the serological diagnosis of infectious disease

Backscattering interferometry enables the detection of syphilis antibody-antigen interactions in the presence of human serum, showing promise as a diagnostic tool for the serological diagnosis of infectious disease with potentially quantitative capabilities.

Backscattering Interferometry for Low Sample Consumption Molecular Interaction Screening

Backscattering interferometry (BSI), which uses a simple optical train comprising a He—Ne laser, a microfluidic channel, and a position sensor, has now enabled the measurement of both tethered and free-solution, label-free, molecular interactions within just nanoliters of sample. The simple macro-to-micro interface allows for a highly efficient assay work flow, which has been used to interrogate molecular binding interactions between proteins, ions and protein, and small molecules and proteins, with a high dynamic range of dissociation constants ( KD) and unmatched sensitivity. With this technique, the equilibrium KD for several different binding partners was determined, typically using just picomole—micromole quantities of the binding pair at physiologically relevant concentrations.

Measurement of monovalent and polyvalent carbohydrate-lectin binding by back-scattering interferometry.

Carbohydrate-protein binding is important to many areas of biochemistry. Here, backscattering interferometry (BSI) has been shown to be a convenient and sensitive method for obtaining quantitative information about the strengths and selectivities of such interactions. The surfaces of glass microfluidic channels were covalently modified with extravidin, to which biotinylated lectins were subsequently attached by incubation and washing. The binding of unmodified carbohydrates to the resulting avidin-immobilized lectins was monitored by BSI. Dose-response curves that were generated within several minutes and were highly reproducible in multiple wash/measure cycles provided adsorption coefficients that showed mannose to bind to concanavalin A (conA) with 3.7 times greater affinity than glucose consistent with literature values. Galactose was observed to bind selectively and with similar affinity to the lectin BS-1. The avidities of polyvalent sugar-coated virus particles for immobilized conA were much higher than monovalent glycans, with increases of 60-200 fold per glycan when arrayed on the exterior surface of cowpea mosaic virus or bacteriophage Qbeta. Sugar-functionalized PAMAM dendrimers showed size-dependent adsorption, which was consistent with the expected density of lectins on the surface. The sensitivity of BSI matches or exceeds that of surface plasmon resonance and quartz crystal microbalance techniques, and is sensitive to the number of binding events, rather than changes in mass. The operational simplicity and generality of BSI, along with the near-native conditions under which the target binding proteins are immobilized, make BSI an attractive method for the quantitative characterization of the binding functions of lectins and other proteins.

Free-solution label-free detection of alpha-crystallin chaperone interactions by back-scattering interferometry.

We report the quantitative, label-free analysis of protein-protein interactions in free solution within picoliter volumes using backscatter interferometry (BSI). Changes in the refractive index are measured for solutions introduced on a PDMS microchip allowing determination of forward and reverse rate constants for two-mode binding. Time-dependent BSI traces are directly fit using a global analysis approach to characterize the interaction of the small heat-shock protein alpha-Crystallin with two substrates: destabilized mutants of T4 lysozyme and the in vivo target betaB1-Crystallin. The results recapitulate the selectivity of alphaB-Crystallin differentially binding T4L mutants according to their free energies of unfolding. Furthermore, we demonstrate that an alphaA-Crystallin mutant linked to hereditary cataract has activated binding to betaB1-Crystallin. Binding isotherms obtained from steady-state values of the BSI signal yielded meaningful dissociation constants and establishes BSI as a novel tool for the rapid identification of molecular partners using exceedingly small sample quantities under physiological conditions. This work demonstrates that BSI can be extended to screen libraries of disease-related mutants to quantify changes in affinity and/or kinetics of binding.

Biosensing with backscattering interferometry

Biosensing with backscattering interferometry

Free-Solution, Label-Free Molecular Interactions Studied by Back-Scattering Interferometry

Free-solution, label-free molecular interactions were investigated with back-scattering interferometry in a simple optical train composed of a helium-neon laser, a microfluidic channel, and a position sensor. Molecular binding interactions between proteins, ions and protein, and small molecules and protein, were determined with high dynamic range dissociation constants (Kd spanning six decades) and unmatched sensitivity (picomolar Kd's and detection limits of 10,000s of molecules). With this technique, equilibrium dissociation constants were quantified for protein A and immunoglobulin G, interleukin-2 with its monoclonal antibody, and calmodulin with calcium ion Ca2+, a small molecule inhibitor, the protein calcineurin, and the M13 peptide. The high sensitivity of back-scattering interferometry and small volumes of microfluidics allowed the entire calmodulin assay to be performed with 200 picomoles of solute.

Solution Interactions via Interferometry

Interferometry is one of several techniques that can be used to probe the concentration and binding interactions of biomolecules, provided that the molecule’s binding partner is immobilized on a surface. Bornhop et al . now show that a fairly simple realization of back-scattering interferometry, which makes use of microfluidic channels for sample mixing and as the multipath cell for the laser light, can be used to determine dissociation constants for several pairs, apparently through changes in refractive index upon binding. Dissociation constants were determined for several binding pairs, including protein A with immunoglobulin G and activated calmodulin (CaM) with a small-molecule inhibitor or with calcineurin. Unlike microcalorimetric methods, these assays can be performed with very low amounts of samples; for example, the several CaM assays required only 200 picomoles of this protein. D. J. Bornhop, J. C. Latham, A. Kussrow, D. A. Markov, R. D. Jones, H. S. Sørensen, Free-solution, label-free molecular interactions studied by back-scattering interferometry. Science 317 , 1732-1736 (2007). [Abstract] [Full Text]

Dual-Capillary Backscatter Interferometry for High-Sensitivity Nanoliter-Volume Refractive Index Detection with Density Gradient Compensation

A simple, stable, ultrasensitive dual-capillary dual-bicell (DCDB) microinterferometic backscattering detection (MIBD) system was developed. In DCDB MIBD, a He-Ne laser beam passes through a half-wave plate onto the cross section of two capillaries, one for reference and another for sensing analyte. The position of the backscattered fringe from each capillary, which are in proximity or essentially identical thermal environments, was detected with matched bicell photodetectors. The configuration was found to effectively compensate for thermal drift, which is normally the major source of noise in refractive index (RI) detection systems. It is shown that passive environmental compensation leads to greatly enhanced signal in nanoscale refractometry preformed by MIBD. An order of magnitude improvement in detection limits over single channel configurations is possible. Performance reaches the 10(-9) RIU level for like solvents in the presence in extremely large thermally induced RI gradients. At this level of detectability, DCDB MIBD could facilitate nanoliter-volume, femtomole-level universal detection in applications ranging from mu-HPLC and on-chip CE to scanning microcalorimetry.

Label-Free Molecular Interaction Determinations with Nanoscale Interferometry

Quantification of protein-protein and ligand-substrate interactions is central to understanding basic cellular function and for evaluating therapeutics. To mimic biological conditions, such studies are best executed without modifying the proteins or ligands (i.e., label-free). While tools for label-free assays exist, they have limitations making them difficult to fully integrate into microfluidic devices. Furthermore, it has been problematic to reduce detection volumes for on-channel universal analyte quantification without compromising sensitivity, as needed in label-free methods. Here we show how backscattering interferometry in rectangular channels (BIRC) facilitates label-free studies within picoliter volumes. The simple and unique optical train was based on rectangular microfluidic channels molded in poly(dimethylsiloxane) and low-power coherent radiation. Quantification of irreversible streptavidin-biotin binding and reversible protein A-human IgG Fc molecular interactions in a 225 pL detection volume was carried out label-free and noninvasively. Detection limits of 47 x 10(-15) mol of biotin reacted with surface-immobilized streptavidin were achieved. In the case of reversible interactions of protein A and the Fc fragment of human IgG, detection limits were determined to be 2 x 10(-15) mol of IgG Fc. These experiments demonstrate for the first time that (1) high-sensitivity universal solute quantification is possible using interferometry performed within micrometer-sized channels formed in inexpensive PDMS chips, (2) label-free reversible molecular interaction can be studied with femtomoles of solute, and (3) BIRC has the potential to quantify binding affinities in a high-throughput format.

Noninvasive fluid flow measurements in microfluidic channels with backscatter interferometry

The ability to measure fluid velocity within picoliter volumes or on-chip noninvasively, is important toward fully realizing the potential of microfluidics and micrototal analysis systems, particularly in applications such as micro-high-performance liquid chromatography (HPLC) or in metering mixing where the flow rate must be quantified. Additionally, these measurements need to be performed directly on moving fluids in a noninvasive fashion. We presented here the proof of principle experiments showing nonintrusive fluid flow measurements can be accomplished on-chip using a pump and probe configuration with backscattering interferometry. The on-chip interferometric backscatter detector (OCIBD) is based on a fiber-coupled HeNe laser that illuminates a portion of an isotropically etched 40 microm radius channel and a position sensitive transducer to measure fringe pattern shifts. An infrared laser with a mechanical shutter is used to heat a section of a flowing volume and the resulting refractive index (RI) change is detected with the OCIBD downstream as a time-dependent RI perturbation. Fluid velocity is quantified as changes in the phase difference between the shutter signal and the OCIBD detected signal in the Fourier domain. The experiments are performed in the range of 3-6 microL/h with 3sigma detection limits determined to be 0.127 nL/s. Additionally, the RI response of the system is calibrated using temperature changes as well as glycerol solutions.

Noninvasive picoliter volume thermometry based on backscatter interferometry.

Using the on-chip refractive index (RI) detector based on backscatter interferometry, sensitive, small volume, noninvasive thermometry can be performed. The current optical configuration for the on-chip interferometric backscatter detector (OCIBD) is quite simple and consists of an unfocused laser, an unaltered chip with a hemispherical channel and a photodetector. Alignment is straightforward with the only requirement being that the beam fully fills the channel. The interaction of an unfocused laser beam with the uncoated etched channel with a curvature within the silica plate (chip) produces fringes whose positional changes scale with respect to the refractive index (RI), n, of the fluid in the channel. Due to the inherently high value of dn/dT for most fluids and the high sensitivity of OCIBD to RI changes, the measurement of small temperature variations in sub-nanoliter volumes is possible. Performing OCIBD with a 75 microm diameter laser beam on a silica chip that contains an etched channel with a 40 microm radius facilitates noninvasive thermometry on a N-(2-hydroxyethyl)piperazine-(2-ethanesulfonic acid) (HEPES) solution in a 188 x 10(-12) L probe volume with a temperature resolution of 9.9 x 10(-4) degrees C, at the 99% confidence level.

Chip-Scale Universal Detection Based on Backscatter Interferometry

An on-chip detector based on backscatter interferometry has been developed to perform subnanoliter-volume refractive index measurements. The detection system consists of a simple, folded optical train based on the interaction of a laser beam and an etched channel, consisting of two radii joined by a flat portion, thus defining a curved surface in the shape of a hemisphere in a silica (glass) plate. The backscattered light from the channel takes on the form of a high-contrast interference pattern that contains information related to the bulk properties of the fluid contained within the probe volume. Positional changes of the interference pattern (fringes) allow for the determination of deltan at the 10(-6) level, corresponding to 743 microM or 139 x 10(-15) mol or 12.8 x 10(-12) g of sucrose, in a probe volume of only 188 x 10(-12) L. A theoretical model of the on-chip backscatter interferometric detector has also been developed, evaluated, and found to be in agreement with experimental data. It is shown that the model can be used to predict general system performance for changes in the optical train such as the chip's wall thickness and channel diameter.

A chip-scale universal detector for electrophoresis based on backscattering interferometry.

An on-chip detector based on backscatter interferometry has been developed to perform sub-nanoliter volume refractive index measurements. The detection system consists of a simple, folded optical train based on the interaction of a laser beam and an etched channel in a silica (glass) plate. This etched channel is composed of two radii joined by a flat portion which define a curved surface in the shape of a half cylinder in a silica (glass) plate. The backscattered light from the channel takes on the form of a high contrast interference pattern that contains information related to the bulk properties of the fluid located within the probe volume. Positional changes of the interference pattern extrema (fringes) allow for the determination of refractive index changes at the 10(-6) level in a detection volume of 188 x 10(-12) L. Under capillary electrophoresis (CE) conditions, the injected mass detection limits for small molecules with little native absorption ranges from 530 fmol (0.18 ng) for sucrose to 720 fmol (0.43 nanograms) for raffinose. Fluorescein was also used to evaluate the technique for universal CE and under further optimized conditions can be quantified at the 150 microM level. Separation performance for the solutes tested ranged from about 2300 to 15,500 plates or 61,000 to 400,000 N m-1. The results presented here indicate there is potential for using the simple optical train of backscattering interferometry for on-chip universal solute analysis.