Bio-layer interferometry

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Figure 1 - Overview schematic of a Bio-layer interferometry setup Bio-layer interferometry without analyte binding.gif
Figure 1 - Overview schematic of a Bio-layer interferometry setup
Figure 2 - The ligand-analyte layer creates an optical path length difference, reflecting incident light in two different patterns Thin film interference - soap bubble.gif
Figure 2 - The ligand-analyte layer creates an optical path length difference, reflecting incident light in two different patterns

Bio-layer interferometry (BLI) is an optical biosensing technology that analyzes biomolecular interactions in real-time without the need for fluorescent labeling. [1] Alongside Surface Plasmon Resonance, BLI is one of few widely available label-free biosensing technologies, a detection style that yields more information in less time than traditional processes. [2] The technology relies on the phase shift-wavelength correlation created between interference patterns off of two unique surfaces on the tip of a biosensor. [3] BLI has significant applications in quantifying binding strength, measuring protein interactions, and identifying properties of reaction kinetics, such as rate constants and reaction rates. [4]

Contents

Method

Mechanism overview

Figure 3 - Reflectance signal as a function of wavelength Bio-layer interferometry wavelength shift due to analyte binding.gif
Figure 3 - Reflectance signal as a function of wavelength

Bio-layer interferometry measures kinetics and biomolecular interactions on a basis of wave interference. To prepare for BLI analysis between two unique biomolecules, the ligand is first immobilized onto a bio compatible biosensor while the analyte is in solution. [5] Shortly after this, the biosensor tip is dipped into the solution and the target molecule will begin to associate with the analyte, producing a layer on top of the biosensor tip. This creates two separate surfaces: the substrate itself, and the substrate interacting with the molecule immobilized on the biosensor tip. [1] This essentially creates a thin-film interference, in which the created layer acts as a thin film bound by these two surfaces. White light from a tungsten lamp is shone onto the biosensor tip and reflected off both surfaces, creating two unique reflection patterns with different intensities. [5] Figure 2 expresses this phenomenon in a more general form. The wavelength shift (Δλ) between these two reflection patterns creates an interference pattern (Figure 3) from which all desired results can be obtained. [1] Since the wavelength shift is direct measure of the change in thickness of the biological layer and the biological layer thickness will change in response to molecules associating to and dissociating from the biosensor, the interference pattern will allow for real-time monitoring of molecular interactions on the biosensor surface. [6] In short, a positive wavelength shift implies an increase in biolayer thickness and thus more association, while a negative wavelength shift implies a decrease in biolayer thickness and thus more dissociation. [6]

"Dip and read" format

Bio-layer interferometry platforms achieve high throughput by utilizing a "Dip and Read" format. [1] The biosensor tips themselves are transported directly to the desired sample and "dipped" into their respective compartment, eliminating the needs for micro-fluidics and the complications (clogging, purification) that come with it. [1] [7] This structure is often supported by a robot, and both 96-well and 384-well plate formats are combined to achieve this. [8] This distinct detection method ensures that sample concentration and viscosity and varying refractive indexes rarely affect the results of BLI. [1] Thus, BLI finds significant use in viscous media such as glycerol, where other techniques may struggle. [9]

Biosensor type and selection

Bio-layer interferometry relies on biosensors with a fiber optic tip upon which the ligand is immobilized. [1] The tip is additionally coated with a matrix biocompatible with the target molecule to limit any non-specific binding. For BLI calculations to work, it is necessary to assume that both the fiber optic tip and the bound ligand and analyte act as thin, reflective surfaces. [10] The biosensors are disposable, resulting in low costs and high commercial availability. [11] Biosensor selection is determined by the desired test results: kinetic analysis, quantitative analysis, or both. [12] Most commercially available biosensor types will be grouped into one of these three categories by the BLI manufacturer. [1]

Applications

Analyzing biomolecular interactions

A key use of Bio-layer interferometry is to analyze and quantify interactions between sets of biomolecules. [1] This is extremely useful in pharmaceutical research, in which biomolecule-membrane interaction determines characteristics of a given drug. Due to its ability to achieve high-resolution data and high throughput, BLI has been used to identify biophysical properties of lipid bilayers, allowing for an alternative method of study than the traditional in vitro methods currently used (microscopy, electrophoresis). [6] In addition, BLI can be used to study effector complex-target interactions. Where the traditional Electrophoretic Mobility Shift Assay (EMSA) method can be used, BLI can act as a suitable substitute if the provided benefits (label-free, real-time measurements) are desired. [3]

Figure 4 - Overview schematic of Surface Plasmon Resonance Surface Plasmon Resonance (SPR).jpg
Figure 4 - Overview schematic of Surface Plasmon Resonance

Measuring biomolecular kinetics

Bio-layer interferometry can be used to analyze kinetics in biomolecular systems. The benefits that BLI brings provide additional insight into kinetics on top of commonly used endpoint methods like enzyme-linked immunosorbent assay (ELISA). [1] Interference patterns found in BLI experiments can be used to calculate rate constants and other kinetic data in biomolecular interactions. [13] The (relatively) lower sensitivity of the BLI sensor results in less response to changes in sample composition. As a result, BLI can also be used to investigate allosteric effects on enzyme conformational changes. [14]

Distinguishing characteristics

BLI and SPR are both dominant technologies in the label-free instruments market. [1] Despite sharing some similarities in concept, there are significant differences between the two techniques. Micro-fluidic SPR relies on a closed architecture to transport samples to a stationary sensor chip (Figure 4). BLI instead utilizes an open system, shaking multiple wells on a plate to transport the sensors to the samples without need for micro-fluidics. [6] Being a closed system, SPR's association and dissociation phases are limited by the technology's design. BLI's open plate design results in association and dissociation length limits determined by sample evaporation instead. [15] SPR is easily reproducible due to its continuous flow microfluidics. BLI's multi well plate design allows for extremely high throughput in one batch. Assay configuration in BLI can, in stable conditions, allow for recovery of samples. Assay configuration in SPR allows for higher sensitivity. As a result, BLI results are often compared to SPR results for validation. [16]

See also

Related Research Articles

A biosensor is an analytical device, used for the detection of a chemical substance, that combines a biological component with a physicochemical detector. The sensitive biological element, e.g. tissue, microorganisms, organelles, cell receptors, enzymes, antibodies, nucleic acids, etc., is a biologically derived material or biomimetic component that interacts with, binds with, or recognizes the analyte under study. The biologically sensitive elements can also be created by biological engineering. The transducer or the detector element, which transforms one signal into another one, works in a physicochemical way: optical, piezoelectric, electrochemical, electrochemiluminescence etc., resulting from the interaction of the analyte with the biological element, to easily measure and quantify. The biosensor reader device connects with the associated electronics or signal processors that are primarily responsible for the display of the results in a user-friendly way. This sometimes accounts for the most expensive part of the sensor device, however it is possible to generate a user friendly display that includes transducer and sensitive element. The readers are usually custom-designed and manufactured to suit the different working principles of biosensors.

Plate readers, also known as microplate readers or microplate photometers, are instruments which are used to detect biological, chemical or physical events of samples in microtiter plates. They are widely used in research, drug discovery, bioassay validation, quality control and manufacturing processes in the pharmaceutical and biotechnological industry and academic organizations. Sample reactions can be assayed in 1-1536 well format microtiter plates. The most common microplate format used in academic research laboratories or clinical diagnostic laboratories is 96-well with a typical reaction volume between 100 and 200 µL per well. Higher density microplates are typically used for screening applications, when throughput and assay cost per sample become critical parameters, with a typical assay volume between 5 and 50 µL per well. Common detection modes for microplate assays are absorbance, fluorescence intensity, luminescence, time-resolved fluorescence, and fluorescence polarization.

A protein microarray is a high-throughput method used to track the interactions and activities of proteins, and to determine their function, and determining function on a large scale. Its main advantage lies in the fact that large numbers of proteins can be tracked in parallel. The chip consists of a support surface such as a glass slide, nitrocellulose membrane, bead, or microtitre plate, to which an array of capture proteins is bound. Probe molecules, typically labeled with a fluorescent dye, are added to the array. Any reaction between the probe and the immobilised protein emits a fluorescent signal that is read by a laser scanner. Protein microarrays are rapid, automated, economical, and highly sensitive, consuming small quantities of samples and reagents. The concept and methodology of protein microarrays was first introduced and illustrated in antibody microarrays in 1983 in a scientific publication and a series of patents. The high-throughput technology behind the protein microarray was relatively easy to develop since it is based on the technology developed for DNA microarrays, which have become the most widely used microarrays.

<span class="mw-page-title-main">Surface plasmon resonance</span> Physical phenomenon of electron resonance

Surface plasmon resonance (SPR) is a phenomenon that occurs where electrons in a thin metal sheet become excited by light that is directed to the sheet with a particular angle of incidence, and then travel parallel to the sheet. Assuming a constant light source wavelength and that the metal sheet is thin, the angle of incidence that triggers SPR is related to the refractive index of the material and even a small change in the refractive index will cause SPR to not be observed. This makes SPR a possible technique for detecting particular substances (analytes) and SPR biosensors have been developed to detect various important biomarkers.

Biacore was a life science products company based in Sweden. In June 2006 Biacore was sold for $390 million and became a product brand under GE Healthcare life Sciences, which became Cytiva in April 2020.

<span class="mw-page-title-main">Enzyme assay</span> Laboratory method for measuring enzymatic activity

Enzyme assays are laboratory methods for measuring enzymatic activity. They are vital for the study of enzyme kinetics and enzyme inhibition.

Dual-polarization interferometry (DPI) is an analytical technique that probes molecular layers adsorbed to the surface of a waveguide using the evanescent wave of a laser beam. It is used to measure the conformational change in proteins, or other biomolecules, as they function.

Nucleic acid methods are the techniques used to study nucleic acids: DNA and RNA.

There are many methods to investigate protein–protein interactions which are the physical contacts of high specificity established between two or more protein molecules involving electrostatic forces and hydrophobic effects. Each of the approaches has its own strengths and weaknesses, especially with regard to the sensitivity and specificity of the method. A high sensitivity means that many of the interactions that occur are detected by the screen. A high specificity indicates that most of the interactions detected by the screen are occurring in reality.

Interferometric reflectance imaging sensor (IRIS), formerly known as the spectral reflectance imaging biosensor (SRIB), is a system that can be used as a biosensing platform capable of high-throughput multiplexing of protein–protein, protein–DNA, and DNA–DNA interactions without the use of any fluorescent labels. The sensing surface is prepared by robotic spotting of biological probes that are immobilized on functionalized Si/SiO2 substrates. IRIS is capable of quantifying biomolecular mass accumulated on the surface.

A thermal shift assay (TSA) measures changes in the thermal denaturation temperature and hence stability of a protein under varying conditions such as variations in drug concentration, buffer pH or ionic strength, redox potential, or sequence mutation. The most common method for measuring protein thermal shifts is differential scanning fluorimetry (DSF) or thermofluor, which utilizes specialized fluorogenic dyes.

A ligand binding assay (LBA) is an assay, or an analytic procedure, which relies on the binding of ligand molecules to receptors, antibodies or other macromolecules. A detection method is used to determine the presence and extent of the ligand-receptor complexes formed, and this is usually determined electrochemically or through a fluorescence detection method. This type of analytic test can be used to test for the presence of target molecules in a sample that are known to bind to the receptor.

An electro-switchable biosurface is a biosensor that is based on an electrode to which a layer of biomolecules has been tethered. An alternating or fixed electrical potential is applied to the electrode which causes changes in the structure and position (movement) of the charged biomolecules. The biosensor is used in science, e.g. biomedical and biophysical research or drug discovery, to assess interactions between biomolecules and binding kinetics as well as changes in size or conformation of biomolecules.

Surface plasmon resonance microscopy (SPRM), also called surface plasmon resonance imaging (SPRI), is a label free analytical tool that combines the surface plasmon resonance of metallic surfaces with imaging of the metallic surface. The heterogeneity of the refractive index of the metallic surface imparts high contrast images, caused by the shift in the resonance angle. SPRM can achieve a sub-nanometer thickness sensitivity and lateral resolution achieves values of micrometer scale. SPRM is used to characterize surfaces such as self-assembled monolayers, multilayer films, metal nanoparticles, oligonucleotide arrays, and binding and reduction reactions. Surface plasmon polaritons are surface electromagnetic waves coupled to oscillating free electrons of a metallic surface that propagate along a metal/dielectric interface. Since polaritons are highly sensitive to small changes in the refractive index of the metallic material, it can be used as a biosensing tool that does not require labeling. SPRM measurements can be made in real-time, such as measuring binding kinetics of membrane proteins in single cells, or DNA hybridization.

Multi-parametric surface plasmon resonance (MP-SPR) is based on surface plasmon resonance (SPR), an established real-time label-free method for biomolecular interaction analysis, but it uses a different optical setup, a goniometric SPR configuration. While MP-SPR provides same kinetic information as SPR, it provides also structural information. Hence, MP-SPR measures both surface interactions and nanolayer properties.

<span class="mw-page-title-main">Single colour reflectometry</span>

Single colour reflectometry (SCORE), formerly known as imaging Reflectometric Interferometry (iRIf) and 1-lambda Reflectometry, is a physical method based on interference of monochromatic light at thin films, which is used to investigate (bio-)molecular interactions. The obtained binding curves using SCORE provide detailed information on kinetics and thermodynamics of the observed interaction(s) as well as on concentrations of the used analytes. These data can be relevant for pharmaceutical screening and drug design, biosensors and other biomedical applications, diagnostics, and cell-based assays.

Brian T. Cunningham is an American engineer, researcher and academic. He is a Donald Biggar Willett Professor of Engineering at University of Illinois at Urbana-Champaign. He is a professor of Electrical and Computer Engineering, and a professor of Bioengineering.

<span class="mw-page-title-main">Kinetic exclusion assay</span>

A kinetic exclusion assay (KinExA) is a type of bioassay in which a solution containing receptor, ligand, and receptor-ligand complex is briefly exposed to additional ligand immobilized on a solid phase.

Focal molography is a biophysical method for robust and sensitive detection of biomolecular interactions in a label-free manner. The new method enables biomolecular interaction analysis in complex biological samples without the use of additional fluorescent labels. Molography widens the analytic scope of biomolecular interaction analysis techniques in a broad range of applications, e.g. label-free trace analysis of a targeted molecule in complex samples, such as blood sera, bioreactor fluid or cell culture media. Contrary to refractometric methods for label-free biomolecular interaction analysis, such as surface plasmon resonance (SPR) and reflectometric interference spectroscopy (RIfS), molography allows quantification of molecular interactions in living cells in real time.

Grating-coupled interferometry (GCI) is a biophysical characterization method mainly used in biochemistry and drug discovery for label-free analysis of molecular interactions. Similar to other optical methods such as surface plasmon resonance (SPR) or bio-layer interferometry (BLI), it is based on measuring refractive index changes within an evanescent field near a sensor surface. After immobilizing a target to the sensor surface, analyte molecules in solution which bind to that target cause a small increase in local refractive index. By monitoring these refractive changes over time characteristics such as kinetic rates and affinity constants of the analyte-target binding, or analyte concentrations, can be determined.

References

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