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{{Short description|Optical biosensing technology}}
[[File:Bio-layer interferometry without analyte binding.gif|thumb|Figure 1 - Overview schematic of a Bio-layer interferometry setup|
== Method ==
=== Mechanism overview ===
[[File:Bio-layer interferometry wavelength shift due to analyte binding.gif|thumb|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.<ref name=":22">{{cite journal | vauthors = Müller-Esparza H, Osorio-Valeriano M, Steube N, Thanbichler M, Randau L | title = Bio-Layer Interferometry Analysis of the Target Binding Activity of CRISPR-Cas Effector Complexes | journal = Frontiers in Molecular Biosciences | volume = 7 | pages = 98 | date = 2020-05-27 | pmid = 32528975 | doi = 10.3389/fmolb.2020.00098 | pmc = 7266957 | doi-access = free }}</ref> 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.<ref name="Apiyo_2017" /> 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 [[Luminous intensity|intensities]].<ref name=":22" /> 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.<ref name="Apiyo_2017" /> 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.<ref name=":13">{{cite journal | vauthors = Wallner J, Lhota G, Jeschek D, Mader A, Vorauer-Uhl K | title = Application of Bio-Layer Interferometry for the analysis of protein/liposome interactions | journal = Journal of Pharmaceutical and Biomedical Analysis | volume = 72 | pages = 150–154 | date = January 2013 | pmid = 23146240 | doi = 10.1016/j.jpba.2012.10.008 }}</ref> 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.<ref name=":13" />
=== "Dip and read" format ===
Bio-layer interferometry platforms achieve high throughput by utilizing a "Dip and Read" format.<ref name="Apiyo_2017" /> 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.<ref name="Apiyo_2017" /><ref>{{cite journal | vauthors = Kamat V, Rafique A | title = Designing binding kinetic assay on the bio-layer interferometry (BLI) biosensor to characterize antibody-antigen interactions | journal = Analytical Biochemistry | volume = 536 | pages = 16–31 | date = November 2017 | pmid = 28802648 | doi = 10.1016/j.ab.2017.08.002 | doi-access = free }}</ref> This structure is often supported by a robot, and both 96-well and 384-well plate formats are combined to achieve this.<ref name=":12">{{cite journal | vauthors = Petersen RL | title = Strategies Using Bio-Layer Interferometry Biosensor Technology for Vaccine Research and Development | journal = Biosensors | volume = 7 | issue = 4 | pages = 49 | date = October 2017 | pmid = 29088096 | doi = 10.3390/bios7040049 | pmc = 5746772 | doi-access = free }}</ref> This distinct detection method ensures that sample concentration and viscosity and varying refractive indexes rarely affect the results of BLI.<ref name="Apiyo_2017" /> Thus, BLI finds significant use in viscous media such as glycerol, where other techniques may struggle.<ref name="pmid27505032">{{cite journal | vauthors = Lea WA, O'Neil PT, Machen AJ, Naik S, Chaudhri T, McGinn-Straub W, Tischer A, Auton MT, Burns JR, Baldwin MR, Khar KR, Karanicolas J, Fisher MT | title = Chaperonin-Based Biolayer Interferometry To Assess the Kinetic Stability of Metastable, Aggregation-Prone Proteins | journal = Biochemistry | volume = 55 | issue = 35 | pages = 4885–908 | date = September 2016 | pmid = 27505032 | pmc = 5524994 | doi = 10.1021/acs.biochem.6b00293 }}</ref>
=== Biosensor type and selection ===
Bio-layer interferometry relies on [[Biosensor|biosensors]] with a fiber optic tip upon which the ligand is immobilized.<ref name="Apiyo_2017" /> 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.<ref>{{Cite journal| vauthors = Gao S, Zheng X, Wu J |date=2017|title=A biolayer interferometry-based competitive biosensor for rapid and sensitive detection of saxitoxin |journal=Sensors and Actuators B: Chemical|volume=246|pages=169–174|doi=10.1016/j.snb.2017.02.078|bibcode=2017SeAcB.246..169G |issn=0925-4005}}</ref> The biosensors are disposable, resulting in low costs and high commercial availability.<ref>{{cite journal | vauthors = Abdiche Y, Malashock D, Pinkerton A, Pons J | title = Determining kinetics and affinities of protein interactions using a parallel real-time label-free biosensor, the Octet | journal = Analytical Biochemistry | volume = 377 | issue = 2 | pages = 209–217 | date = June 2008 | pmid = 18405656 | doi = 10.1016/j.ab.2008.03.035 | doi-access = free }}</ref> Biosensor selection is determined by the desired test results: kinetic analysis, quantitative analysis, or both.<ref>{{cite journal | vauthors = Yu Y, Mitchell S, Lynaugh H, Brown M, Nobrega RP, Zhi X, Sun T, Caffry I, Cao Y, Yang R, Burnina I, Xu Y, Estep P | display-authors = 6 | title = Understanding ForteBio's Sensors for High-Throughput Kinetic and Epitope Screening for Purified Antibodies and Yeast Culture Supernatant | journal = Journal of Biomolecular Screening | volume = 21 | issue = 1 | pages = 88–95 | date = January 2016 | pmid = 26442912 | doi = 10.1177/1087057115609564 | pmc = 4708621 | doi-access = free }}</ref> Most commercially available biosensor types will be grouped into one of these three categories by the BLI manufacturer.<ref name="Apiyo_2017" />
== Applications ==
=== Analyzing
A key use of Bio-layer interferometry is to analyze and quantify interactions between sets of biomolecules.<ref name="
=== Measuring
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 [[ELISA|enzyme-linked immunosorbent assay]] (ELISA).<ref name="
BLI and SPR are both dominant technologies in the label-free instruments market.<ref name="
==See also==
* [[Interference reflection microscopy]]
* [[Surface plasmon resonance microscopy]]
▲== Distinguishing Characteristics ==
▲BLI and SPR are both dominant technologies in the label-free instruments market.<ref name=":0" /> 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. BLI instead utilizes an open system, shaking multiple wells on a plate to transport the sensors to the samples without need for [[Microfluidics|micro-fluidics]].<ref name=":2">{{Cite journal|last1=Wallner|first1=Jakob|last2=Lhota|first2=Gabriele|last3=Jeschek|first3=Dominik|last4=Mader|first4=Alexander|last5=Vorauer-Uhl|first5=Karola|date=2013|title=Application of Bio-Layer Interferometry for the analysis of protein/liposome interactions|url=http://dx.doi.org/10.1016/j.jpba.2012.10.008|journal=Journal of Pharmaceutical and Biomedical Analysis|volume=72|pages=150–154|doi=10.1016/j.jpba.2012.10.008|pmid=23146240|issn=0731-7085}}</ref> 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.<ref>{{Cite journal|last1=Abdiche|first1=Yasmina|last2=Malashock|first2=Dan|last3=Pinkerton|first3=Alanna|last4=Pons|first4=Jaume|date=2008|title=Determining kinetics and affinities of protein interactions using a parallel real-time label-free biosensor, the Octet|url=http://dx.doi.org/10.1016/j.ab.2008.03.035|journal=Analytical Biochemistry|volume=377|issue=2|pages=209–217|doi=10.1016/j.ab.2008.03.035|pmid=18405656|issn=0003-2697}}</ref> 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.<ref>{{Cite journal|last1=Yang|first1=Danlin|last2=Singh|first2=Ajit|last3=Wu|first3=Helen|last4=Kroe-Barrett|first4=Rachel|date=2016|title=Comparison of biosensor platforms in the evaluation of high affinity antibody-antigen binding kinetics|url=http://dx.doi.org/10.1016/j.ab.2016.06.024|journal=Analytical Biochemistry|volume=508|pages=78–96|doi=10.1016/j.ab.2016.06.024|pmid=27365220|issn=0003-2697}}</ref>
== References ==
{{Reflist|2}}
[[Category:Biosensors]]
[[Category:Molecular biology techniques]]
[[Category:Interferometry]]
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