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after the interaction of drug the enhancement in the SPR band was observed the intewraction was proved by FTIR and AFM the AFM resiults shows aggregation of NPs the of NPS increases from 40nm to 200 studied bty AFM i also study time depedent interaction of drug and nanoparticles the DLS spectra showing abrupt results how to interpret the results.
DLS result attached plz guide
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Check out this website:
Get your results analyzed by an expert quickly! The service is not free but it's very affordable and definitely worth it.
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I did some SPR experiments in another lab, but now that I am back in my own lab we do not have a biacore instrument and thus neither the software. Can anyone please recommend a software to just view the results files? I do not need to produce any data, I just want to be able to see the result file with the responses and the time. Thanks !
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Julian Plaga : Dropped you a note. I do need to extract the blr files.
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I am searching for the main question "why we need to study SPR"
1. Is commercially available SPR devices are not giving enough sensitivity?
2. What is the need of SPR market for improvement?
3. main objective to study SPR
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In SPR , Kretschmann and Raether configuration use the Prism to couple the incident p-plorized light to the sensor system. This can be achive with the help of glass slab too. what is the advantage of prism geometry over the rectagular glass slab?
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triangle or semicylindrical prism(s) ensure, that you cross the air to prism boundary at nomal incidence. Thus the polarisation state is not altered.
See for example Fig. 1 of:
and Fig. 1 of:
Taking a rectangular prism, I do not know how you will maintain the polarization state of the incoming laser beam.
You may share your configuration in mind...
Best regards
G.M.
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Hello,
I received this SPR sensogram for small molecule binding affinity test on a protein. Would you please explain to me what are those dotted lined next to solid line? Thanks
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Moustafa, hard to judge since the instrument type used is unknown. The solid lines seem to be the recorded data (including huge buffer shifts), the dotted lines seem to show fitting trials that ignore the buffer shifts. - You should ask the person (from whom you received the data) for details about data recording and fitting parameters.
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Surface Plasmon Resonance (SPR) - in a nano-particle for example, is caused by the collective oscillation of the conduction electrons, causing a distinct optical absorption.
If such a particle is placed in a very strong external electric field (such as in the middle of a MIM capacitor type arrangement) should it not then be possible to cause the static polarisation of these electrons, so that they are no longer free to resonate, effectively stopping the SPR and stopping their optical absorption - making the particle "transparent"?
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Dear all, please search using 'attenuation of Surface Plasmon Resonance', many investigations are available. Please have a look at the following documents, hope they will be usefull somehow. My Regards
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about pml convergence test.
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SPR indicates formation of silver nao in UV-vis spectrophotometer data it gives SPR Peak
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B R Siddharth To know about the size, morphology, concentration, fraction of spherical to nonspherical nanoparticles, and size distribution of NPs from UV-Vis data, the UV-Vis data (especially the surface plasmon resonance) is fitted with Mie model (for spherical NPs) and Mie-Gans model (for nonspherical NPs). The Mie model is based on the Maxwell equations accounting for the discontinuity of the dielectric constant between the metal sphere and the surrounding medium. It has been successfully applied to free and functionalized metal nanoparticles in various solvents or matrices with diameters in the range 4-25 nm. Despite the differences among samples, an accuracy of about 6% on the nanoparticle's average size with respect to sizes measured by transmission electron microscopy (TEM). Moreover, the fitting model provides other information not available from TEM like the fraction of spherical to nonspherical nanoparticles as well as the concentration and size distribution of NPs in the sample, which is particularly useful for measuring aggregation processes.
In the following video tutorial, I have used two Mathematica codes based on the above two models to calculate various properties associated with the NPS. The codes and other relevant material are provided in the video description. Thanks
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Hello All,
I am working on finding out binding kinetics of nanoMIPs against and inactivated virus using SPR. I have observed that with decreasing the flow rate of the sample (4 ul/min->2 ul/min->0 ul/min), the rate of association increases.. which is contrary to what literature (and logic) says if my experiment is suffering from Mass transport limitation. It would be really nice if you can shed a light on this?
I am looking forward to your responses.
Thanks
Best regards :)
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Strange. Pls, provide ass curves pics here,
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I have a question about the SPR (BI-4500) from Biosensing USA. I am trying to adapt an existing method used on the Biacore3000 before to this new instrument. For the Biacore, the flow rate was really low (5- 10 uL/min) while the recommended flow rate for the BI-4500 is 30-60 uL/min. Anyone has done experiments side-by-side with the Biacore and this instrument and got the same results? How do you recommend adjusting the flow rate?
Thanks!
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No idea what kind of interaction you want to do but in general we use a flow-rate of minimal 30 ul/min in the Biacore 3000 and sometimes higher. Mass transport can be lower at higher flow rates but for pure 1:1 kinetics flow rate should not be of influence.
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i'm looking for the right way to compute the dispersion relation related to SPR surface plasmon polaritons using the formula ( Kspp = (w/c)sqrt((eps_d*eps_m)/eps_d+eps_m)
should I consider both real and imaginary parts of the dielectric function (available in matrix form in Matlab) or only the real part, I'll be thankful for your positive responses
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Your question is discussed in chapter 2 of classical textbook of H. Raether "Surface Plasmons on Smooth and Rough Surfaces and on Gratings", Springer-Verlag, Berlin 1988.
Epsilon must be in complex form.
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Typical SPR slides are made of glass with the 50nm gold coating on top. But I wanted to know how I could create SPR slides made from clear flexible plastic with the coating on top so that I can roll it into a cylinder or cut it into pieces? So I was wondering if anyone had done something similar before or knows the process?
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In the past, I did coated my plastic fibers with gold and it worked very well. Let me know if you want to know more.
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I wanted to create a SPR Fiber Optic cable with one side of sensing region exposed and filled with a gold metal layer. I found that many papers used a more elaborate automated wheel polishing setup to grind the cladding down to their desired level. I don't have these setup built so I was looking for an easier approach to removing the cladding.
The easiest method I came across was from the following research paper where they used a polymer coating of a 400 µm (core diameter) plastic cladded silica (PCS) fiber (Thorlabs BFL48-400, NA = 0.48) which was removed first by using a razor blade and then the bare glass fiber was cleaned by using acetone and distilled water subsequently.
I was also thinking of another approach which involved going to the machine shop and milling down the cladding to the desired level. Are there any other ways to create a single sided SPR fiber optic cable?
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It seems that you are using a glass fiber. Don't bother using them: they are too difficult and brittle to work with and breaks very easily. Switch to plastic fibers and you will be able to use them in a far more productive fashion.
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I came across a Fibre Optic SPR sensor used with a phone and I was trying to figure out what thickness they used for their sensing region.
They did not mention the thickness so I went into their references and saw one paper mention that the thickness of the silver coated sensing region on the fibre optic has to be at least 300 nm. Compared to prism SPR its very large probably since we are trying to reflect the signal. But I wanted to be sure about the thickness used for the coating before getting it fabricated.
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I'd be carefully looking at the patents and patent applications. These often contain vital and useful practical information. For example, the attached.
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I have calculated the kd value for peptide (13 mer peptide) and LPS (Lipopolysaccharide) interaction through SPR and fluorescence displacement assay and got 1000 fold difference. the equilibrium dissociation constant value is 15 micromolar in fluorescence titration experiment and 1.3 millimolar (Affinity constant) in SPR experiment . Can anybody suggest why I am getting this difference?
SPR experimental condition
Immobilization buffer-10 mM PBS buffer (pH-6.0)
Running buffer-10 mM HEPES, 150 mM NaCl, pH 7.4)
250 micromolar peptide was immobilized and 49 µM, 98 µM, 245 µM, 490 µM, 980 µM concentrations of LPS were injected and affinitiy constant was calculated for peptide-LPS interaction.
For Fluorescence assay
Tris-cl (pH -7.4) was used
BODIPY-TR-cadaverine (BC) as a fluorescent probe
Increase in fluorescence intensities (at 680 nm wavelength) with increase in concentration of peptides were plotted and the equilbrium dissociation constant (Kd) was calculated using Igor Pro 6.36 (Y = Bmax × Xˆh/(Kdˆh + Xˆh); where X: concentration of peptide (µM), Y: fluorescence intensity (a.u.), h: Hill slope)
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I agree with Vanessa Porkolab.
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I would like to ask questions about how the adsorbed molecular influence frequency and intensity of surface plasmon resonance of metals such as Ag and Au.
Does the adsorption of molecular influence the frequency and intensity of SPR simultaneously?
Or will different adsorption configurations of the same molecular have specific influences? (just for example, linear adsorption will only influence the intensity but bridge adsorption influence frequency and intensity simultaneously?)
Could you recommend some answers or papers (books)? Thank you so much.
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Xu Xuanwen, A well-localized, evanescent SPR field is excited surface absorbed molecules. Then this field is re-emitted due to their own oscillations with a frequency of incident light. These oscillations are specific to the kind of absorbed molecules, their index of refraction, concentration (so, SPR intensity), other optical properties. In such a way, absorbed molecules influence on optical properties of the whole SPR system.
Strong surface localization of the SPR field produces strong sensitivity to only surface absorbed molecules and their state, which can be changed due to some specific reaction. Such effect is the origin of SPR application as chemical, biological sensors of different kinds.
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I am trying to measure the spr effect by making 100 50 10 nm sized silver nanoparticles.
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This effect can be very well characterized by UV-visible (actually in the visible range). For this, the particles must be widely dispersed and in sufficient concentration. From my experience, silver SPR does not look as good as it does on other metals such as gold.
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Hi,
I want to make a simple SPR setup. I have a prism (from a microscope), spr chip from NanoSPR for its glass n=1.61, immersion oil with n=1.515, a red laser pointer, a disposable 3d glasses as a polarizer, and white paper as a screen.
I tried to scan from 0 to 90 degree of incident angle manually. But I couldn't see any minimum. What I missed? What is wrong? Do you have any idea?
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This and following articles exactly related to answer on your question; I try to give it in very concise form, but there you can find complete one
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Which are the main limitations of SPR comparing with BLI, especially in terms of buffer and matrix especially for liposome-protein interactions assays ?
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BLI uses substantially more sample volume (something like 20-40uL in 384well, more like 100-200 in 96well), but like others have said, is *mostly* non-destructive (depending on how robust your sample is to agitation and ambient temperatures). BLI is less sensitive than traditional SPR (not suited to fragment screening and other small molecule apps) but is more amenable to crude samples such as sera/lysates etc since the tips are disposable. It's much faster for high-throughput type apps (especially if you can get your hands on the 96-channel top-of-the-line models), and so potentially much faster. Both systems need some amount of knowledge to be used properly. SPR can do some more complicated and interesting things (e.g. enzyme kinetics if you have at least a 4-surface consecutive flow system) and generally more robust data, but more finicky when it comes to troubleshooting. SPR can also be end-coupled to mass spec in some configurations.
Liposomes is a bit vague - is your protein embedded in the liposomes? or you are looking simply at protein-membrane binding? Either way you could have options with both SPR and BLI but you would need to optimize for your application. My personal recommendation is SPR but it may not be entirely objective ;)
Hope this helps
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Hi all,
I am considering to use SPR and I wondered how much bacterial culture is usually needed in the grow up phase to end up with enough sample having sufficient protein concentration? There might not be a definite answer since protein expression can go south in many ways but getting an idea about lower and upper bounds would also help me.
For NMR experiments I always did a 2 litre grow up and most of the time had enough sample. My impression is, that this is not needed for SPR if the protein expresses reasonably well and if there are no issues during the purification.
Looking forward to your answers.
cheers
Martin
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Does absorbance peak in UV-Vis result can be related to the diameter of nanoparticles in SEM image? Because for UV-Vis, the absorbance peak has high intensity at 520nm and which indicates the formation of the gold nanoparticles right? But from SEM image of AuNPs, some of NPs have diameter over 100nm (which from my understanding over100nm are no longer in the nanoparticles range).
#the gold nanoparticles were synthesized using PLAL method#
#gold nanoparticles were red wine color#
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There are good comments above from Leo Vleugels and Omar Z. Sharaf . I would add the following:
  • Gustav Mie based his interpretation of light interacting with matter via Maxwell's equations on the color of gold sols. He has plots within his classic 198 paper for maxima in the VIS region based on the size of the gold nanoparticles. See (registration required): The life of Gustav Mie and the development of the Lorenz-Mie solution to Maxwell’s equations https://www.malvernpanalytical.com/en/learn/events-and-training/webinars/W140225Gustav-mie-maxwell.html
  • Carry out a Stokes' Law equation based on the size and density (19.3 g/cm3) for gold. I think you'll find that 100 nm particles cannot stay in stable suspension
Based on the above 2 bullet points, and if you have a transparent (no turbidity or evidence of settling) purple sol then the likely size for your material is in the ~ 5 - 50 nm region and your particles of > 100 nm shown in SEM are a result of agglomeration and aggregation in the SEM preparation stage (drying , coating etc). Take a look at:
Dispersion and nanotechnology
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Can anybody help me out
1. how to insert power fraction equation in COMSOL while calculating relative sensitivity?
2. What is S(nm/RIU) in Figure of merit formula= S(nm/RIU)/FWHM(nm). Is it wavelength sensitivity or another term?
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Thank you Tahhan sir
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We have a BI4500 SPR, by which we can generate very good data for BSA/anti-BSA antibody and biotin-DNA/cDNA interaction.
Recently we try to use this machine to study ssDNA aptamer/protein interaction. We immobilized protein by CM chip, NTA chip or avidin chip, but can't observe any binding with the aptamer. We also use avidin chip to immobilize the biotin-aptamer, still can't see any binding with the protein. However, we successfully observed good binding if we use flow cytometry or fluorescent imaging for this aptamer and protein. This is very strange!!! Could you pls give me some suggestions?
Thanks.
Shawn
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Good luck, Shawn!
In case you have success, I would like to hear from you.
Yours,
Hans
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i am trying to find KD of aptamer as the analyte and protein. I'm using a CM sensor chip. I'm using PBS with tween 0.005% and 1 mM MgCl2 as RB. Normally I don't use ethanolamine to block the channel because aptamers don't have primary amine that can bind to the activated COO- and also we observed good binding without EA. we thought EA blocks everything and so we didn't use EA. with EA we couldn't generate any results. recently we got a good KD and it was pMol range. so it is too good to be true for this aptamer. when we checking specific binding, we realized there was some mistake in this experiment. so we activated the reference channel and injected the aptamer. we saw the binding, but that is not a non-specific binding. does someone have an idea about yhis? if that please help with this.
we assumed there should be a kind of positive charge interaction and that should be some kind of contaminants of the CM chip. it is a new CM chip. does anyone know any cleaning procedure for CM chip?
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Vanessa,
We generated SA chip by ourselves and successfully immobilize biotin-aptamer on the chip, but still, we can't observe the binding for the target protein.
This is very strange, so we did several control experiments: 1) we successfully observed the interaction between biotin-ssDNA and its cDNA; 2) we confirmed that the aptamer can bind the protein by flow cytometry and fluorescent microscope.
Could you pls give some advice?
Thanks.
XT
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Hello,
I just started reading about plasmonics and I have few doubts. I googled a lot but couldn't find concrete answer.
Please correct me if I'm wrong:
  • Plasmon is quantum of plasma oscillation: Charge density oscillation (free electrons) on any surface [electrons vibrate around their equilibrium positions at certain characteristic frequencies called plasma frequency, which depends only on the number density of electrons, electric charge, electron mass and permittivity of free space].
  • Surface plasmon resonance is the phenomena or event of coupling of EM wave with oscillating conduction electrons of the metal nanofilm.
  • Surface plasmon polariton is the propagating EM wave generated at the metal-dielctric interface that propagates along the surface of the metal film after surface plasmon resonance.
  • But Peter Y. Yu's answer confused me. He said that " To form a SPP, the photon and SPR must have the same frequency and wave vector" in Link: (https://www.researchgate.net/post/Is_there_any_differences_between_Surface_plasmon_polariton_and_surface_plasmon_resonance)
  • How SPR can couple with photon since it is a phenomena?
  • I've seen people mentioning about dipoles, SPR and SPP. What is the relation?
  • In wire grid polarizer TM passes because dipole form , they couple and radiate. How is plasmonic effect really involved in the working of Wire grid polarizer?
Thank you.
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Dear Ambar Shukla , I agree, when one is starting in this field the terms couls be puzzling.
-Not any surface can produce plasmons, you need free electrons to cause the oscillation, so a metal or an electron rich material would be necessary.
-To many and for the sake of simplification SPR and SPP are synonyms, look for instance:
However, I think that Peter Y. Yu answer is more accurate, since you can excite a plasmon polariton with an electromagnetic (EM) wave or with a beam of electrons.
About the relation of plasmons with dipoles, when a metal or plasmonic material is exposed to an EM wave, the electric field polarizes the charges into the metal, causing a dipole (induced by the field). This is often represented with metal nanoparticles with sizes smaller or close to the wavelength, but it is also applicable to surfaces or interfaces, just that in these cases the wave propagation left an alternating charge separation along the surface.
Hope it helps.
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Hello, I have developed a Surface Plasmon resonance sensor using LED of wavelength 635nm and CMOS webcam as source. I am using the diverging rays of the LED as the change in incident angle. When I put silver coated glass slide on the prism I get a dip at a particular angle. I have test the sensor by immobilizing with MUA , EDC/NHS and IgG. The sensor can detect the shift in angle for all the layers. But when I put liquid dielectric medium like DI water, BSA or PBS buffer the shift disappears. I can monitor real-time data with the webcam and so when the liquid sample is passed I should be able to detect the shift. I have attached the file of how the dip looks like.
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Surface plasmon resonance (SPR) slides contain a glass slide with 50nm thick gold coating on top of it. Instead of using the Kretschmann setup with a bulky prism and having to worry about alignment, why do we not use the SPR slides as a waveguide and just shine led at the edge of the slide and have a spectrometer at the other end? Then you place some solution on the gold film and there should be a change in the spectrum. Ensuring everything is covered up so there is not ambient light, what are some issues with this idea or considerations?
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Can anyone please suggest some good reading material/papers on thermal effect of surface plasmon resonance (SPR)? I want to generate approximately 100 degree C with SPR.
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SPR slides contain antibodies that bind to specific target molecules during use. But once the reaction is over, the slide needs to be replaced with a fresh slide with the Au film and antibodies. I was wondering what are the techniques for "refreshing" the slides so it can be used again. I had come across a paper where biotin-streptavidin interaction can be reversibly broken using warm water, but are there any other processes for different ligand-conjugate pairs?
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Hello, everyone.
In SPR or BLI, I wonder if apparent difference of KD (binding affinity) requires comparison.
I heard at least 10-fold comparison of KD means apparent difference.
Then, less 10-fold comparison of KD does not means difference?
Could I see the reference about this?
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Adam B Shapiro I agree more or less with that, but very often statistically significant differences do not reflect significant "size effects".
For example, it may be possible for a 2-fold difference to be statistically significant after 10 replicates, but the 2-fold difference is not physiologically/practically relevant. The same size effect can be made to be statistically relevant just by increasing the number of replicates, and this is something that can be employed to magnify small differences. The abusive use of p-values has put them into question.
On the other hand, Kd's should be averaged employing the geometric average, not the arithmetic average, and, consequently, their geometric standard deviation should also be calculated. This derives from the logarithmic relationship between Kd's and ΔG's (equilibrium constants would follow a log-normal distribution and energies would follow a normal distribution).
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Currently, I am trying to simulate a surface plasmon resonance (SPR) biosensor on a multilayer structure using FDTD Lumerical software. I am a beginner and have never used this software before. Are there any tutorials related to SPR simulation using this software? This document was very meaningful in completing my research.
Thank you
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Hi Devi,
Lumerical software has a bunch of "example" simulation you can try from their fresh installed. The simplest way to start, you call one of this sample, take a look at the simulation design and condition, and how it works then try to modify and to your own case, you can change the material, or change the shape, or size (step by step). Do not forget to "save as.." to the new file name in your common folder.
Then try to run, and you got your first simulation case on Lumerical. If the error appears, you need to trace back from the example file, what was the mistake, then try to fix the error.
Good luck.
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I am currently looking at this SPR spectra of different sizes of AU nanoparticles and I’ve come to realise that there is a absorption area that increases in intensity towards the left of the SPR band. Is it caused by Inter or intraband transitions?
Many thanks,
Fabian
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This article can be help in understanding.
"The Au NPs are characterized by interband transitions at energies
above 2.5 eV, whose tails extend into the surface-plasmon resonance (SPR) region, hence affecting the SPR energy, amplitude, and broadening"
Losurdo M. Size dependence of the dielectric function of silicon-supported
plasmonic gold nanoparticles / M. Losurdo, M.M. Giangregorio, G.V. Bianko et al. //Phys. Rev. B. – 2010. – Vol.82. – P. 155451
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The protein I'm studying is called A.
A can bind to multiple targets, which are identified by indirect ELISA and SPR kinetics analysis.
Most of the target proteins were confirmed to bind in ELISA, and corresponding results were also shown in SPR kinetics analysis.
However, ELISA binding didn't appear only in any one target protein. (But in SPR, it showed good values of ka and kd) Can't ELISA compare to SPR? I can't figure out what's the problem.
1) When looking at ELISA binding, can this be explained in connection with kinetics? (I other words, if the ELISA OD value is high, can it be regarded as having a low KD value?)
2) ELISA binding is not performed, but ins SPR kinetics, how should I interpret the result value? Is this a problem with the htarget protein? (I purchased and used it, but I know that it is very difficult to express and purify. Is it possible that it is the effect of the protein?)
If you are awared of these details, please let me know. Thanks!!:)
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I think a dissosiation rate of 10-2 s-1 and faster is too fast to be detected with ELISA.
From your first post you asked wheter you can compare ELISA readout with SPR-affinity.
In short NO. ELISA endpoint reading is not an affinity reading. If you want to determine the solid state affinity with ELISA you must do a concentration series and plot the readout versus the added protein concentration. You must be sure that the protein concentration is the limiting factor and not the coating or the detection.
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We ran SPR using dextran chips and always obtained very good data. For example, we can clearly discriminate wide type analytes and mutations. Recently, we failed all SPR works and can't repeat any previous experiments. However, we still can detect good SPR signals using BSA and anti-BAS antibody.
Here is an example to study PDL-1 protein/PDL-1-1 aptamer. You may see good activation, protein immobilization, blocking, but just no protein aptamer binding. Again, we did genearate strong signals using the same materials before (the same proteins and DNA aptamers, which are two months old and stored well at -20C). It seems something changes protein conformation since we know antibody still can recognize BSA even it is denatured. Could it be sodium acetate (pH 4.8) or ethanolamine etc? But how and why? Can anyone pls help us?
Thank you very much!
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Protein level after the hPDL1 pulse is much smaller than the sum of the slope during the hPDL1 pulse. In fact, after the ethanolamine pulse the level is essentially back to the level before the hPDL1 pulse. Seems you don't have much stable covalent immobilization of the protein. How about changing the EDC and NHS reagents?
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I am testing binding affinities of various antibodies on amyloid fibrils & oligomers using surface plasmon resonance (SPR). I am unable to regenerate the surface (ie remove the amyloid) using the usual regeneration reagents (HCl, SDS, etc). Any ideas?
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Hi. I use piranha solution to clean of the chip that is covered by biomaterials. But, when I put it in solution all of my gold layer is dissolved in solution.
Do you have any idea about it and what I have to do?
Best sincerely
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From phage diaplay library,I sort an antibody and express it in HEK293F cell line,and I can have the IgG protein,but when I use this IgG to test the kinetics by SPR, I cant see any binding.But use ELISA assay, I can see the protein interaction..
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Depends on the SPR resolution,as you know,the phage is so small. maybe it had bind in SPR surface,but just couldn't detect it.
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How would the spectrometer signal change if the target molecule was well dispersed in a solution and placed on the fiber optic SPR cable VS the target molecule concentrated at a specific region on the fiber optic cable?
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The effect can be very strong. If the concentration change is e.g. aggregation, then not only shielding effect blocks light from reaching molecules inside an aggregate, but also spectra might be changed by interactions between the molecules in the aggregate. In some cases, addition of 0.1% of a solvent which prevents aggregation transforms a clear (for eyes) solution into a deeply colored one, even when no precipitation was seen in the original suspension.
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Does anyone have experience with TGFbeta reconstitution? I want to reconstitute it but the final conditions of the solution should be compatible with my SPR running buffer, which is PBS-P+3%DMSO. I was thinking of reconstituting it in PBS-P 4mMHCl containing an appropriate amount of DMSO. Do you think the buffer (PBS instead of water) will affect the dilution (by affecting the final pH for example)? Will DMSO help? Why use HCl to reconstitute TGFbeta, where does it help?
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Did you ever get an answer for this? I am also looking to reconstitute initially in DMSO, then further serial dilutions in PBS.
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I used COMSOL 5.4 for modeling and simulation. The attachment is my file, but I still don't get a good loss spectrum. I think it's the reason for PML, but I tried a lot of methods and the final result is wrong.
Thank you for your attention!
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Have you got it fine? If so share file
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I am working on a PCF based plasmonic sensor for refractive index detection. The work is simulation based using COMSOL Multiphysics software. Theoretically how can I calculate the efficiency of the coupling between the incident light and plasmonic coupling for 2D model?
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Hello
You can see the details about the coupled-mode theory for the PCF-SPR sensor from the attached paper in section III.
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i am performing SPR experiment and i am referring some journal and websites they stated regarding coupling buffer and immobilization buffer so ihave confusion about these buffer.
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For amine coupling on a carboxylated dextran chip (CM5) the buffer requirement is that it is free of amines. So PBS, HEPES are good buffers, TRIS not.
For more basic answers see www.sprppages.nl
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Farhan Mumtaz Wow! you are based in Wuhan. Hope you're developing sensitive and selective labelesss sensor for COVID-19 virus using this tech. Wonderful tech.!
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I want to use gold nanocluster in my research.
So I try to find the difference between them, but except for size, I don't' know well the exact difference.
on my found information, AuNCs do not exhibit surface plasmon resonance (SPR) absorption in the visible region.
What are the reasons and advantages?
I would like to know the difference between gold nanocluster and gold nanoparticle.
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Gold nanoclusters are favoured by most researchers due to their strong stability in fluorescence and adjustability in fluorescence wavelength when compared to traditional organic fluorescent dyes. For gold nanoparticles with diameters between 3–100 nm, the strong surface plasmon resonance (SPR) dominates in the absorption spectrum, which is caused by collective excitation of free electrons. In contrast, ultra-small gold nanoparticles consisting of tens to hundreds of gold atoms, often called gold nanoclusters, show multiple absorption bands spanning the UV-visible NIR range because of discrete energy levels.
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Hi,
I am studying on polydopamine coated sensor chip for SPR biosensor for my masters thesis research. I am trying to detect BSA with SPR analysis but I could not observe angle shift in every analysis. I use the same polydopamine cotaed chip over and over and, I clean the chip ultrasonically in isopropanol before SPR analysis. May the problem be using the same chip over and over and/or cleaning ultrasonically in the isopropanol?
Thanks!
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Dear Dr. Adam Shapiro,
Thank you for your answer. I viewed this article. Isopropanol, acetone ect. have no significant effect on deattachment of PDA on the sensor surface, including ultrasonication. Nevertheless, I can't observe angle shift even in high concentration of BSA, that is to say proteins don't attach to the sensor surface. But why? I have to solve this problem.
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In all the literature I've read, I always find Au and Ag as metals for surface plasmon resonance photocatalysis. Why are other metals never used?
One option I've considered is that other metals oxidise and cannot remain in metallic form to act as a photocatalyst, but then, why do we never see Pt SPR for example?
Thanks in advance!
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Au and Ag nanoparticles can be formed to absorb visible spectrum efficiently that's why its commonly used photocatalysts.
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Hi,
I am planning on running some protein-protein interaction/kinetics studies using the Nicoya COOH sensors in OpenSPR. I am looking for the activation and blocking buffer composition that are needed for this coupling (EDC/NHS). Anyone here has experience using these sensors from Nicoya? Could you share your detailed protocol and buffers (composition?
-Thanks
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Amine Coupling: A ligand is immobilized onto slide with available carboxylate groups. The carboxylate groups on the surface of the slide are modified with linker, a mixture of EDC and NHS to form an ester. A covalent amide bond is then formed to the ligand which must have at least one free primary amine group. Injection of ethanolamine removes any loosely bound ligand. Analyte is injected over the surface at a range of concentrations higher and lower than the expected equilibrium dissociation constant (K).
Chemicals:
EDC (Sigma Aldrich Cat. No. 03449-5G)
NHS (Pierce Cat No. 24500) 10mM
Sodium acetate pH 5.2 (need pH about 2 units below pI of ligand to be coupled)
1M ethanolamine pH 8.5 (Sigma >98% Cat No. EO135 – you dilute to 1M and pH to 8.5 with HCl)
PBST (Sigma Cat No. P 3563) or whatever buffer is needed
Immobilisation:
Flowrate: 20-50 µL/min
SPR prism temperature: 25 °C 4.
Steps:
Weigh 40 mg EDC and 10 mg NHS into a suitable vial and dilute with 0.9 mL of water. Dissolve and inject as quickly as possible after diluting. Inject over both channels for 7 minutes 20 µL/min.
Prepare a solution of 30-50 µg/mL of proteine in 10mM sodium acetate pH 5.2 buffer. Over the sample channel only, inject for about 10 minutes or until a maximum has been achieved.
Inject over both channels 1M ethanolamine HCl for 10 minutes (blocked the unspecific binding sites).
Inject the solution of the ligand (other protein) for both channel, than remove the excess amount by running buffer.
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The buffer is already supplemented with 0.05% Tween 20 and 1% BSA.
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Try using CM4 chip, use ethlyene diamine acetate instead of ethanolamine and also run carboxylmethyldextran with the analyte. They are sold as NSB reducer from GE. This should greatly reduce the binding of analyte to the sensor chip.
Hope this helps
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I am investigating the Surface Plasmon Resonance (SPR) of metal nanoparticles, namely gold and silver particles. If you know anything about it or have an in-depth article on it, please share it with me, I appreciate it.
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I want to investigate protein interaction by SPR. I want to do amine coupling for ligand immobilization on-chip surface. first, activation of the surface by NHS/EDC, and then injection of the ligand should be done. based on chemistry, activation of carboxymethyl surface by NHS/EDC should be done at pH 4.5-7.2, and the reaction of NHS-activated molecules with primary amines of the ligand is most efficient in pH7-8 (Ref: Thermo scientific), but in the SPR instrument book (Biocare) mention ligand immobilization by amine coupling should be done at pH 1 unit lower than the isoelectric point of ligand due to electrostatic interaction toward the carboxymethylated surface. I get confused! which pH should be set for ligand immobilization?!
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Dear Parastou, I agree with Michael. The Thermo protocol may be the best from a purely chemical coupling point of view. The Biacore protocol favours concentration enrichment of protein ligand close to the surface. Simply try a few different pH values according to the different protocols, and most likely you'll find something that works well. Best, Anders
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In a SPR experiment with Biacore T200, I'd like to immobilize my amine-functionalized ssDNA ligands (around 80mer) on the CM5 chip. Although I changed pH , I couldn't immobilize the ligands on the chip well in my first try. I used phosphate buffer (6.5-8) for immobilization. I sincerely appreciate it if you give me your knowledge for this kind of matter.
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You can try the biotin labeled ssDNA to immobilize on the SA chip.
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what is a proper protocol i can follow for an experiment regarding nano fabrication of hollow structures in SPR? , for example the methods and the instruments needed, chemical solutions, etc, and thanks.
Yours sincerely,
Saif
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Porous nanomaterials are synthesized by a sol-gel method (metal and non-metal oxides) or an electrochemical method. Porous silicon is obtained by electrochemical method in the presence of hydrofluoric acid. Surface plasmon resonance appears when electromagnetic radiation interacts with a collection of electrons (plasmons) on the surface of nanoparticles of gold, silver ... Therefore, it is necessary to synthesize a hollow structure, and then attach gold nanoparticles in the cavity.
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Hi,
I am looking for substantial evidence of effect of geomerty of flow channels (microfluidics) on the sensitivity and feasibility of Surface Plasmon Resonance (SPR) Biosensors with respect to different applications (whole cell, protein or DNA detection). I know one from Prof. Jiri Homola.
I need to design my own microfluidic channels for SPR sensing applications. Please guide me.
Thanks :)
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I am trying to evaluate and analyze SPR data in TraceDrawer software. But when I try to analyze my data, the software cannot process it, the prompt saying that .txt file is not recognised
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Thanks, I did,
this is the reply I received-
"Hi, Ahmed,
I assume that you have a TraceDrawer license key that enables you to open text files and that you acquired this key from one of the companies to which we provide the software. Usually, these companies give direct support to their customers."
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Why does Selenium nano particles exhibit SPR absorption which is not a metal? looking for for explanation and literature
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One possibility, which I have heard in other cases is that, while Se is not a metal, light excitation can generate a free electron population high enough to give a negative dielectric constant, end hence the appearance of a SPR.
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I am looking into the indirect immobilisation of CXCR4 onto the sensor chip surface in SPR experiments. Previous work looking at this has involved engineering the receptor so that it has a C9 tag (TETSQVAPA) on the C-terminus. 
My question is, why would this be used in favour of more widely used tags such as GST or Histidine, for which there are capture kits available for Biacore Biosensor technology? 
It is not a tag I have come across until now.
Any help would be great
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Do you get the answer why use the C9 tag?
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I Want to evaluate the binding affinity characterization and kinetic binding of the antibody coated on the nanoparticle.
SPR, Blitz based experiments could help?
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Hello,
As a newbie to SPR, I've been having problems with optimizing aptamer-protein binding on an NTA chip. My protein has a his-tag on it. Any help would be greatly appreciated.
Question 1:
Do the running buffer, analyte buffer and ligand buffer all need to match? ie. The running buffer in the start-up cycle is the same as the buffer to dilute the aptamer analyte and the protein ligand to appropriate concentrations.
Question 2:
My running buffer is: 25 mM Tris-HCl pH7.5, 100 mM NaCl, 20 mM imidazole, 0.005% Tween 20 and 0.5 mg/mL BSA. When I run a startup cycle, the injection of running buffer results in a noticeable increase in RU.
I have also tried: 10 mM HEPES, 150 mM NaCl, 50 µM EDTA. 0.005% Tween 20, pH 7.4. With same results. I read that running buffer should not increase the RU, how can I solve this issue?
Question 3:
As a result when I inject analyte (diluted in running buffer) it also results in an increase in RU. But because I've matched running buffer, analyte and ligand, the resulting Fc=2-1 is flat, with small short dip and peak at injection times. Is this an accepted result?
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Question 1:
Do the running buffer, analyte buffer and ligand buffer all need to match?
Yes perfect match to avoid the bulk effect. The best way is to equilibrate your sensorchip in 20 min of running buffer. Then, you can start a start up experiment and the analyte buffer will be your running buffer. For your ligand buffer, it will be the running buffer.
Question 2:
The injection of solution on FC1 and FC2 give you a signal. Normal. You have to check the subtraction of FC2-FC1 to see if the signal is close to 0.
Question 3:
The aptamer has to be diluted in the running buffer. After subtraction FC2-FC1, if you don't see increasing signal, it means that there is no binding. However, aptamers are difficult to work with using SPR. They are negatively charged and the Carboxydextran CM sensorchips are also negatively charged. So, you could have an electrostatic repulsion while you are injecting your analyte in solution. I will recommend you to capture the aptamer with a biotin. And then, inject your protein.
Vanessa
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I am planning to perform an antibody-ligand interaction study with SPR/Biacore. The idea is to immobilize my antibody with Protein A/G. It would be great if you could share your comments, what to consider, advantages/disadvantages of Protein A/G immobilization for SPR.
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I recommend you to buy the protein A sensor chip instead to made your own protein A sensor chip. Because some signal drift could be observed after the amine-coupling of protein A. The protein A is commonly used compared to protein G.
For my personal experience, I prefer to use the igG kit or Fab kit using a regular CM5 sensor chip. Using this kit you are not worry about the regeneration buffer and these kits show reproducible data.
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I need some clarification about SPR and LSPR.. 
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I'd also get confused about these two concepts once. Sadly no answer helped - wikipedia, quora and researchgate or many papers/books given. They tended to be full of a loose combination of random terminologies.
Finally I understood once I found a review paper: 'Surface plasmon resonance in gold nanoparticles: a review', Figure 2 is very helpful (I guess you just want to understand the difference in a figurative rather than fully precise or mathematical way.)
SPR is surely a more generic term and can be divided into propogating SPR (occuring on a bulk metal surface) and localised SPR (occuring on a metal nanoparticle surface). Now the key points are the difference between 'propogating' and localised' and why. Localised SPR literally means the phenomenon is limited in space, because very importantly, the nanoparticle is so small that is even much smaller than the wavelength of visible light. So the free electron gas density can only dance (or oscillate, why? please think with the explanation below) collectively as a whole on that particle surface, and resonate with the part of the light wave with the right frequency.
By contrast, for bulk metals, (ideally) when the light wave is moving parallel to the plane of the metal-air (for example) interface, propagating SPR occurs on the surface which is very large in dimensions. This results in regionally and moving resonance of the free electron gas density along the propogating direction of the light wave. To further understand this, it is important to recall that light is in nature alternating, propogating magnetic and electric fields. The electric field therefore indicates alternatingly positive and negative potentials along the propogating dimension just above the metal surface, consequently inducing alternatingly negative and positive potentials on the metal surface. These regularly ever-changing induced potential differences then leads to back-and-forth movement of the free electron gas density, which is namely oscillation and with the right frequency, resonance. And because the light is propogating, so the regional motion has also to propogate along.
So that you can see, as an interesting consequence just because of the differences in the dimensions of the interface, localised SPR is transverse whereas propogating SPR is longitudinal, with respect to the light wave.
Hope this explains clearly.
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I have designed a SPR chip by using normal glass slide(Blue star No. 4slide)/Cu(5nm)/Ag(50nm). But it shows very less depth in the reflectance curve at resonance angle. What should be the reason ??
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Thank you Jonathan Shilantha Weerakkody for sharing your expertise on the fabrication of SPR-based devices, especially regarding that pesky adhesion layer! A major challenge (i.e. hot topic for research) is to have a mechanically stable adhesion layer which does not deteriorate the plasmons at the interface.
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  • I am working on ssssynthesis of gold nanostructures
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The answer is simple, as the other responses have explained, the SPR, in the case of nanoparticles the localized SPR, produces a strong enhancement of the electric field on the nanoparticles surface. The Raman effect is a nonlinear effect which strongly depends on the electric field strength, therefore if the material being analyzed is attached to the NP surface, the Raman effect is greatly enhanced, hence SERS. And yes, using a spelling corrector is a good idea...
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We are looking at whether a peptide: RNA as a complex can bind to a glycosylated protein. We have established with SPR that the Peptide binds to RNA. Is it possible to study the binding of the peptide:RNA complex to a third binding partner with SPR. The experiment I plan to do is to immobilize the third binding partner i.e. the glycosylated protein on the SPR sensor chip and then sequentially flow and dissociate 1) RNA 2) Peptide and 3) RNA+ Peptide mix. Has any one done these kind of interactions with SPR. I am using the Open SPR machine form Nicoya lide
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Hi ! you can easily do it but you need to identify which binding partners have a low koff rate when they are interacting together, i.e. a slow dissociation phase has to be observed
For example, you could immobilize the peptide or the RNA onto your sensor chip (at high density). Then, you inject your second partner with no dissociation time (slow dissociation observed). At this stage, you are able to inject your third partner. If the 3nrd partner doesn't compete with the second one, you should observe a binding. And you can repeat the experiment at different concentrations and you should be able to get a KD. That's the principle of the epitope binning.
Good luck Nikhil Kulkarni
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Can the Biacore detect binding of an analyte which is mostly or completely enveloped by the ligand binding pocket upon association?
Since SPR measures the thickness of the sensor's surface, and binding of an analyte as described above would theoretically result in no net change of surface thickness, is it possible for the Biacore or any other SPR platform to detect this binding phenomenon?
For context, I've previously run multicycle kinetics of the ligand and complete analyte with good results. I tried to run a multicycle kinetics with the same ligand and a specific moiety of the same analyte and did not detetct any binding. The literature indicates that the moiety is the epitope bound by the ligand and computer modeling would indicate that the moiety would be almost entirely enveloped by the binding pocket of the ligand. 
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Hi, Biacore detects change in SPR angle in real time which changes when mass change happens on the sensor surface causing change of refractive index on the surface. So if any molecule is interacting, it should be shifting the SPR angle irrespective of the confirmation change.
you should run the buffer scouting and regeneration scouting protocols and find optimum conditions for the assay.
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I would like to know if there is a quantitative justification for using a specific threshold to calculate steady state Kd affinity using surface plasmon resonance (SPR). Some people say 10% but I don't know that this is backed by any data or simulations.
My goal is to be able to establish a cutoff for which we can confidently calculate steady state Kd for weak interactions. For example, you have a protein-protein interaction that has a Kd of 200uM and the highest you can go due to analyte supply is 2uM, then you won’t reach saturation. However, you can still fit the isotherm to get a Kd steady state value. In this case the final response will be much lower than the fitted Rmax.
Do you know of any papers that look into assigning a Kd value based on the noise in the system and possibly some simulations? For example, is a cutoff of 10% (final response over predicted Rmax) sufficient to assign a steady state Kd based on any models?
Thanks
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@Rm Mal it has been recommended to keep the Rmax around 100-150 for reliable kD if the evaluation software is being used. However, the minimum Rmax I am not sure about but I think the noise level is around 2 RU when in running buffer - and may vary depending on buffer type..
Anyway, 15 Rmax has been reported before and see no reason why 10 RU should be reliable.. Of course the higher the better as long as there is no mass transport and the signal to noise ratio is reasonable to get a signal to fit the data..
Question : Can't you immobilise the analyte and use the ligand as "analyte" if the ligand has a bigger mass to increase the RU...?
By the way to get a realistic KD try 10 times less and 10 times more than the KD for the analyte concentrations for the binding..
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Hello,
as it is commonly stated in literature (spheric) silver, gold and copper NPs exhibit absorption around 450 - 600 nm. It is possible to tune this absoprtion wavelength by shape modification. But barely I am finding information about NP absorption of other elements/metals. Whats the reason for that?
I am trying to produce gold-NP with an absorption around 800-1200 nm and get a high density of these nanorods. I am encountering several problems, e.g. stick them close to the surface of my samples and still have them plasmonic active and avoid aggregation. So I was thinking about other elements:
Is there a metal/element that shows NP absorption in the near IR region (800-1200 nm)?
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Gold and silver are the "gold standard" for plasmonics. Hence, most articles involving plasmonic absorption use these two noble metals.
As Omar Z. Sharaf highlighted, the extinction spectra is adjustable via geometry manipulation. Take a look at this paper
Alternatively, it might be better to utilise a plasmonic film (as opposed to nanoparticles) depending on your application.
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Hi.
I plan to analyze binding affinity with SPR (biacore).
I want to use NTA sensor chip because my ligand-protein (10 kD; no Lys residue)has 6xHis tag.
But the problem is that the analyte protein (70 kD) has 6xHis too.
So after ligand immobilization, I want to block the remained Ni-NTA functional group (which is not binding with ligand).
This is my step by step protocol for SPR analysis.
1. Activate NTA sensor chip with Ni.
2. Immobilize the His tagged ligand.
3. block the chip. ------> this is waht i want to know
4. inject the analyte with various concentration.
Actually, when I asked about it to GE healthcare technical support team of our city, She said How about change the sensor chip to CM5 and change the position of ligand and analyte.
Because of my 10 kD His tagged peptide has no Lys residue, I cannot immobilize it on CM5 chip with amine coupling reaction.
I asked about L-Histidine for blocking agent too, but she said "The interaction between monomer histidine and Ni-NTA is too weak to remain on the chip during the experiments".
So, I think how about use polyhistidine (sigma, MW 5,000~25,000) for blocking reagent.
But I wonder if the polyhistidine is used as a blocking reagent, the immobilized 10 kD His tagged ligand is remain or competition reaction with polyhistidine.
Is there anyone who did like me?
Thank you.
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Thank you for your reply.
I'm sorry to late.
I tried to use the methods that..
1. Immobilized the His-tqgged peptide as a ligand
2. block with poly-Histidine
3. inject analyte
I could get a binding sensorgram, however, the ligand was removed from the chip because of the regeneration solution (Gly-HCl 3.0 or EDTA included soln. or NaOH).
The immobilized His-tagged peptide was removed from the chip because of the regeneration soln., and whenever the different concentration of the analyte was injected, I had to immobilize the His-tagged peptide again.
It was a time-consuming method and it is difficult to maintain the orientation of the immobilized peptide.
So, We changed to used CM5 chip rather than NTA chip.
1. immobilize the peptide with amine coupling as a ligand
2. blocking with EA (mentioned above)
3. inject analyte
Thank you for your interest :)
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I want to design a Surface Plasmon Resonance Chipset. But I don't know how to design the thickness of the glass under the gold film. And I wnat to know if the thickness of the glass can affect SPR
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Hello Oleh Yermakov , Thanks for your answer.
I have some questions.
I study SPR structures for depositing metal on optical fiber surfaces, which can be seen as Kretschmann configurations?
If so, in the SPR structure using optical fiber, if the thickness of the fiber becomes thick, can it be interpreted to mean that the ratio of Dip is reduced?
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I should carry out a set of SPR experiments.
In my lab I have the Reichert model "SR7500DC".
I would need someone who could teach me how use this instrument and, above all, how to do the data interpretation.
Could anyone help me, please?
Thank you very much
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Dear Emanuele,
you can call/email us anytime if you need any assistance with the instrument/data evaluation.
Best regards,
Nico
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Surface plasmon resonance of silver nano particles synthesis
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In continuation with Rina Singh , the LSPR peak for silver nano particles vary from ultra violet to violet region of spectrum depending on its size and shape. However for gold nano particles it is 510 nm onwards again depending on its shape and size.
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We have bought the SPR Reichert SR7500DC to investigate the binding between an antibody and the AG protein blocked on the chip.
Please, could anyone tell me how this istrument work?
Thanks
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Hi Jules Lloyd Hammond,
sorry if I'm replying you now, but I got forced to leave the instrument right now.
Could you indicate me the name of the lab where you used this instrument, or whoever could use this specific instrument, please?
Thank a lot
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Hi,
I am trying to differentiate small molecules based on their KD values using SPR. My small molecules are very tight binder (non-dissociator), hence have very low Kd & KD values (pM - fM). However their is come some difference in Ka values. Can I rank order them on Ka values? Is their any other way i can differentiate between them.
Appreciate your inputs.
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You can use an experimental setup known as kinetic titration, or single-cycle kinetic analysis for binding partners with too high affinity to feasibly regenerate the ligand surface.
Briefly, 5 increasing concentrations of analyte are injected in series over the ligand surface for identical contact times, with a short dissociation between each, followed by a long dissociation period. You can determine the interaction rate constants from this data and thus the interaction equilibrium constant. BiaEval software has analysis models for kinetic titrations out of the box, and you can modify equations from https://www.sprpages.nl/data-fitting/models or the reference below as needed.
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Good morning,
I've heard that results obtained with Ni-NTA sensor chip in SPR are seen as less accurate than with amine coupling from the experts in the field. Is it true? If so, why?
Thank you!
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To obtain a more stable bond between the His6 ligand and the sensor chip surface, so-called poly-NTA sensor chips can also be used: https://www.xantec.com/news/whitepapers_newsletters_nihc-high_affinity_poly-ni-nta_sensor_chips_for_fragment_based_drug_discovery.php
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its colorimetric detection after interacting a analye with nanoparticles color rapidly change from yellow to orange. after 15 min of interaction take of a absorption spectra only quenching (decrease in absorption intensity) was observed large nanoaggregates was formed which confrmed by DLS. after 45 min visible aggregates are seen in solution. why shift not appear ?
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Dear Ayesha Urooj,
Given that chemical and physical structural changes in light absorbing entities under observation may either cause the absorption maxima of observed spectral absorption bands to move up or down the visible spectrum, or alter their spectral bandwidth, I reason as follows.
The term SPR {Spectral Plasmon Resonance) refers to the mode of vibrational displacement of free (un-bonded) electrons either within a given molecule’s chemical structure, or within a more loosely bonded molecular aggregation.
Three rules now follow: (1) the larger the free path of the electron within the structure the lower the oscillation frequency and therefore, the longer the mean wavelength of light absorption maximum. (2) If more than one resonating entity (each with a distinct frequency) is present peak broadening is observed. (3) Any given resonance mode may easily be inhibited or even quashed by introducing additional resonation modes.
I suggest firstly that in terms of colour and at the low molecular count associated with nano-particles, they behave as ‘super molecules’. Thus, in the initial change from yellow to orange small two and three molecule aggregations are generated which exhibit slightly longer electron resonance path. As aggregation proceeds the subsequent quenching and peak broadening indicate that there is a rapid rise in both quantity and variety of contributing vibrational modes and frequencies.
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I am interested in its sensitivity , sample consumption and overall performance. Thank you.
No advertising for SPR or other instruments please. I am only interested in the Octet and BLI.
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Thank you!
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If you need to filter 500 ul of a protein that´s in a buffer with 1% DMSO, how would you filter this before putting in an analytical equipment, like for SPR? Would you use regenerated cellulose syringe filter?
In our case, we could add the DMSO to the protein after filtering through a low protein binding filter. This is convenient, but then the DMSO won´t be filtered.
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