Questions related to SPR
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
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 !
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
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?
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
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"?
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.
Best regards :)
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?
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
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?
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?
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.
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)
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.
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?
Which are the main limitations of SPR comparing with BLI, especially in terms of buffer and matrix especially for liposome-protein interactions assays ?
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.
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#
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?
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?
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?
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.
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?
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?
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?
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.
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?
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!!:)
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!
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?
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..
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?
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?
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!
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?
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.
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.
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?
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!
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?
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.
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?!
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.
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.
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.
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
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
I Want to evaluate the binding affinity characterization and kinetic binding of the antibody coated on the nanoparticle.
SPR, Blitz based experiments could help?
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.
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.
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?
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?
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.
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 ??
- I am working on ssssynthesis of gold nanostructures
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
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.
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?
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)?
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?
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
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
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.
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?
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 ?
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.
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.