Immobilization - Science topic

It isn't surprising that the nanoparticle-adsorbed enzyme has lower activity than the enzyme in solution. In fact, this is precisely the mechanism that was proposed about 20 years ago by McGovern and Shoichet to explain why some chemical compounds behave as non-specific enzyme inhibitors. They showed that some compounds form nanoparticles that remain suspended in solution, and adsorption of the enzyme to these nanoparticles inhibits the enzyme. Presumably, this is because adsorption causes a structural change in the enzyme, or blocks the active site, or prevents the enzyme from undergoing a necessary conformational change involved in catalysis.

In order to avoid this sort of nonspecific inhibition, you should look for a way to immobilize the enzyme by a flexible linker that allows it freedom of movement.

Using immobilization and running buffers at pH 5.5 in Surface Plasmon Resonance (SPR) can have some potential disadvantages.

It is important to consider these potential disadvantages and to test your protein-antibiotic interactions at a range of pH values to determine if pH 5.5 is the optimal condition for your specific system. Additionally, It is also important to check if the immobilization buffer and running buffer that you are using are compatible with the pH you are using.

References:

Thank you soo much for your response sir Mohamed Hassan

I need this paper

"PASTORE, M., MORISI, F., VIGLIA, A. International Symposium Mario

Negri Institute. Milan, 15-16 June, 1973."

can you help me?

You can run the reaction with an immobilized enzyme if you can keep the nanoparticles fully suspended during the reaction, then pellet them quickly (either by centrifugation or with a magnet) and measure product formation in the supernatant. One way to do this is to perform the reactions in microcentrifuge tubes that fit into an Eppendorf Thermomixer (or similar instrument). If the nanoparticles are small enough, they may even stay in suspension well enough without mixing, as long as the reaction time is not too long.

Biochar amendment is a promising approach to mitigate soil contamination via immobilizing heavy metals and organic pollutants. The quality characteristics of biochar as a soil amendment varied greatly with the feedstock materials and the pyrolysis conditions. Soil-incorporated biochar was able to stabilize Cd, Cu, Ni, Pb, and Zn and reduce their bioavailability through enhanced sorption and chemical precipitation. The stabilization efficacy was largely determined by cation exchange capacity, pH, and ash content of the biochar. Biochar amendment increased the mobility of anionic toxic elements in soil. Soil-incorporated biochar was also able to absorb non-polar organic compounds and polar organic compounds. The adsorption efficiency was controlled by the biochar surface properties specific surface area, microporosity, and hydrophobicity. Biochar may facilitate the mineralization of organic pollutants by enhancing soil microbial activities. The effectiveness of biochar-facilitated soil remediation was case specific, changing with the biochar source, amendment rate, placement, soil type, and pollutant species. Biochar reduces soil density and soil hardening, increases soil aeration and cation-exchange capacity, and changes the soil structure and consistency through the changes in physical and chemical properties. It also helps to reclaim degraded soils.The larger surface area and higher surface energy are helpful for biochars to strongly absorb the heavy metal pollutants and remove them from the soil. Possible mechanisms for the heavy metal retention by chars are the formation of metal (hydr) oxide, carbonate, or phosphate precipitates and/or the activation of surfaces caused by the pH rise, and sorptive interactions between d-electrons of metals and aromatic π-electrons of chars, and specific metal.

Experiment this technique:

1. Dialyze the sample against a suitable KBr solution

2. Expose the sample to evaporation under vacuum at low temperature

I just found people use ethanolamine in the SA coating.

Enzymes are polypeptides . The Change in pH effects the ionisation state of the side chains of amino acids. The charge on enzyme protein changes.

square wave voltammetry

Prof. Annwyne Houldsworth: Please let me ask the following three questions:

Quiero hacer especial énfasis, en que si bien la ansiedad patológica o psiquiátrica, debe ser diagnosticada por el buen clínico (pues la psiquiatría es una especialidad clínica), su diagnóstico positivo lleva de la mano el diagnóstico diferencial de las entidades orgánicas que cursan con ansiedad, que de no ser diagnosticadas a su debido tiempo, pueden pasar inadvertidas y, ocasionar discapacidad, cronificación de enfermedades, discapacidad y hasta la muerte, de ahí, el valor semiológico que tiene hacer el diagnóstico de un cuadro ansioso, frecuente en el ejercicio de la práctica médica de cualquier especialidad.

a) Enfermedades endocrinas. Entre ellas destacan: 1) hiper e hipotiroidismo, que pueden presentarse inicialmente sólo con signos y síntomas de ansiedad, en ocasiones persistentes, aunque se compense la alteración tiroidea; 2) feocromocitoma, cuyas crisis se manifiestan con cefalea intensa, sudoración, enrojecimiento e hipertensión arterial; 3) hipoglucemia; 4) hiperparatiroidismo, que se confirma determinando niveles de calcio sérico; y 5) enfermedad de Cushing.

b) Enfermedades cardiopulmonares. Destacan: insuficiencia cardíaca congestiva, prolapso de válvula mitral, embolismo pulmonar, arritmias (p. ej., taquicardia supraventricular paroxística), enfermedad pulmonar obstructiva crónica (EPOC), asma bronquial, apnea del sueño, neumonía e hiperventilación. En ocasiones puede confundirse un episodio de angor péctoris con una crisis de angustia, de forma que es conveniente la realización de un ECG.

c) Enfermedades neurológicas. Entre ellas se distinguen: crisis comiciales parciales complejas, accidentes cerebrovasculares (de córtex frontal, insular o temporolímbico), tumores del tercer ventrículo, encefalitis, enfermedades desmielinizantes, enfermedad de Wilson y trastornos vestibulares.

d) Enfermedades metabólicas (déficit de vitamina B12, porfiria aguda intermitente, acidosis metabólica, entre otras.).

e) Otras (síndrome carcinoide, enfermedades del colágeno, brucelosis y otros).

f) Trastorno de ansiedad inducido por sustancias (cafeína, anfetaminas, corticoides, cocaína, atebrina, quinacrina, insecticidas, epinefrina, aminofilina, teofilina, drogas de abuso sobre todo estimulantes del sistema nervioso central).

ELISA technique can be adapted for measuring the antibody against polysaccharides of bacterial or other origin. However, to my knowledge, it cannot be adapted for a direct determination of polysaccharides.

I have been trying to do that only. Thank you Cory Clark , this was very much helpful.

I few have few knowledge about PDMS optical coating layers !

Thank you.

Please refer below review paper,it will be surely answered you,

Hi,

First, you should use a bigger bottle, if you incubate 6g in 40ml a 50 ml Bottle is to small and you can´t expect aerobic conditions. I would suggest to use 100 ml Duranbottles.

Second, don´t shake the nitrifiers so much. It is better just to use 100 rpm

Third, the initial NH4 concentration should not be higher than 1mM.

Forth, your incubations should be longer, up to one week. Depending on soil type and temperature.

For you Ammonium, Nitrite and Nitrate measurements: I don´t know the SmartChem method. Maybe you should use the classical photometric ones.

To distinguish between net und gross nitrification, labelling with 15N nitrate is a possible method.

Best,

Tina

Kindly check the reference

Biochemical Engineering Journal 1998 2 (3) 179-186

Max J. Stein , yes, it does show up in discussions of social mobility (immobility) but in migration - it is often approached as what people DON'T do, and while there are links between mobility and immobility, NOT migrating is not the same as being immobile, it has to be more than a physical movement defined by a relationship to a geographic point in time as both Nicolas Parent and Neema Ghenim argue. At an institutional level, if immobility is referenced as NOT mobility, we lose the complexities that drive immobility as a household decision for example.

While Fe³⁺ is a stable configuration, catalytic activity is still possible. Whether it is actually happening in your case, is a whole different beast of a question.

The immobilization efficiency should be calculated with respect to the surface of the particles. The same is true for the concentration of glutaraldehyde. Are you quantifying the concentration of amine groups on the surface? You could estimate your efficiency using this concentration.

Dear Divya Jose thanks for sharing this very interesting technical question with the RG community. Personally I'm not an expert in this field, but I just came across the following potentially useful review article which should help you in your analysis:

This review has been published Open Access so that it is freely available as public full text (please see attached pdf file).

I hope this helps. Good luck with your work and best wishes!

Good morning Tarun Gangar.

What you seek is basically KNOWLEDGE about...

Fortunately,in this time and age KNOWLEDGE in any field is easily accessible on the Internet.Just key in the major words of the subject and then,presto!!!- you get the answers ~,response.

My caution and guide to you is to verify the source of the information to make sure that it is solid ,reliable creditable etc.

Hoping this helps out?

Dear all, the following documents give detailled experimental procedure in doing characterization of nanoparticles, and the information obtained via each technique. My Regards

10.1039/9781782621867-00001

My concern is that i dont wanna use any chemical as crosslinker, I want to use polymer.

Dear Komal Grover, better to use the term 'adsorption' than 'immobilization'. In the following review, the mechanism of interaction is discussed. My Regards

Thank you Prof. Majid, When we compare different electrodes modified step by step and the ks value differs than what can be interpreted from that data?

I agree that indeed there can be a set of intermolecular interactions between CuO and CNT functionalities. But, it seems, the degree of interactions strongly depend on conditions of your precipitation method. If it was a typical precipitation from the copper salt by alkali in the presence of the dispersed CNTs followed by calcination at high temperature, then you really oxidized the CNTs and could see this change in their composition by FTIR. To make sure in the interaction you need to compare FTIR spectra of the mechanical mixture of CuO and CNTs and the nanocomposite. Besides, if you really used calcination you could calcinate pure CNTs under the same conditions as the nanocomposite and to make their spectrum after this treatment.

Hey Kasturi!

Because some impurities have an affinity to nickel columns - some proteins just happen to have a few surface histidines in close proximity to each other, and they will bind to your column. Further, your protein might also happen to bind to some host proteins that get co-eluted. Generally, you should wash your column with a buffer that contains a low concentration of imidazole (if your protein elutes at 20mM, maybe try washing with 5mM). Further, you can pool your step elutions if they are clean enough and you can concentrate them using something like an Amicon centrifugation concentrator. I would recommend that you then perform a size exclusion chromatography if you urgently need protein that clean for your assays.

Hello Marius,

When i need to count motile green algae such as Dunaliella or Tetraselmis i use a diluted solution of iodine tincture, just few drops in your sample, but don't exceed or you will have a too bright color.

Otherwise you can prepare the classic lugol solution or a 5% formaldehyde solution

I concur with Jordan T White that using log-scale would give a better readout of the actual binding affinities. The differences in the layout of the two plots makes comparison of the values difficult.

I would determine the 'linear range' of the upper plot to be between 30 - 250 ng/ml, while the 'linear range' of the lower plot is <5 ng/ml -< 50 ng/ml; above 50 ng/ml, it is non-linear. But just looking at the two plots, the upper one, being sigmoidal, suggest co-operative binding, while the lower one looks like a saturation curve.

Perhaps there is a valency difference in the two proteins? I know that Fc chimeras will form dimerize to form bivalent chains. I do not know if Fc-Tag chimeras dimerize or not. I am unfamilar with that platform.

Dear Yin Li thank you for your kind response and explanation. In that case my suggestion would be to search the "Publications" section of RG for relevant research terms related to your question. It is very important that you limit your search to the most meaningful terms. Here are the search results for "copper-based biosensors" in the "Publications" section:

For example, please have a look at the following potentially useful article:

This paper is freely available as public full text on RG.

Also please have a look at the following interesting link:

Dear Warda Mustfa thank you for asking this interesting technical question.As an inorganic chemist I'm certainly not an expert in this field. However, I found a few relevant references which might help you in your analysis. For example, please go through the following useful articles in which the use of animal-based biochar is reported:

Immobilization of Lead and Cadmium in Soil Using Biochars Derived from Pig Manure and Suaeda glauca

and

(see attached pdf file)

I hope this helps. Good luck with your work! 👍

Phosphate buffer is not a good option for buffering the alginate beads since phosphate itself have a strong affinity to Ca ions bound to alginate. The initial problem might also have arisen from this fact; because the initial media also had the phosphate buffer (mineral salt media is also buffered with phosphate) the beads decay through time. A good alternative is to use non-phosphate buffers for pH control, Good's buffers are good for it (We utilized MOPS for pH 7).

For more info on immobilization see Bickerstaff's book:

Sorry I saw the question 4 years late, maybe it helps for other people.

Have a nice research!

Faezeh Mohammadi Please read the following book (chapter 3; link provided below) for detailed explanation of the proposed mechanisms of action.

It probably depends on the coating quality you need. If it is not principal you can try a pipettor with a suitable volume (e.g. 0.1-2.5 microliter) to drop a small portion of the solution.

Please look at the following below links which may help you in your analysis.

Thanks

Eduardo Cueto Diaz

The nanoparticles will rapidly dissolve in HF; the borosilicate will be much more resistant especially as it's under the Au. So you have 2 choices:

What have you got to lose? Other than your health - proper safeguards for HF use are essential. All that glisters is not gold...

April 1, 2021

Dear Usama,

Knowing the amount of protein immobilized on the Dowex is not the most important parameter as some of the enzyme protein may have been inactivated during the immobilization process. So you need to measure the activity, not the amount of bound protein.

One approach would be to determine the dry wt of the Dowex/enzyme complex, e.g., mg/mL Dowex/enzyme suspension. For the enzyme assay, add an accurately-measured volume of suspension to your buffer, followed by the substrate. It is important to keep the resin/enzyme in suspension, so incubate with stirring (very small stir bar) at a suitable temperature. At periodic intervals (e.g., 0.5- or 1 min), withdraw measured volumes of the liquid over time and measure the amount of product formed/min. This can be used to calculate the # enzyme units/mL (or per dry wt) of the Dowex/enzyme complex.

I hope this information helps you.

Bill Colonna Ames, Iowa, USA williamjcolonna6@gmail.com

Have a look here:

There are different projects for the immobliziation of "nano"mof on differnet surfaces. A group in Dresden Germany did some work, but i dont know anymore which group it had been.

Yes, I'm using it now for testing GOx immobilized on an electrode with an outer membrane. I centrifuged my electrodes (0.5mm dia wire x ~4mm long) in 100uL of the assay buffer for 10min at 5.2G. Then I transfer to a plate well and add 50uL of the reaction mix. I'm seeing the absorbance on the plate reader increase on all measurements from about 0.04 up to about .4 over about an hour.

I would suggest diluting your GOx in the assay buffer until it falls within the linear range which according to the kit is between 0 and 0.6.

Uday Kumar Banala All the best in your research. It will be a challenge to selectively adsorb uranium from such matrix.

I guess i was reading one paper,

The link is here with which answers your question i suppose

You may add 5 % of acrylic acid to your acrylamide. Subsequently, the polymer can be activated by a carbodiimide/NHS mixture.

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

Thank you very much for your help, I will check it!

The specific activity of an enzyme preparation is the turnover-number (molecules of substrate that an enzyme molecule handles per second) multiplied with the enzyme concentration.

What your reviewer is in effect asking is

Any such effect could invalidate your observed K_m. However, I am not sure that this belongs into the introduction. Evidence of full activity should be presented under results, and its significance in the context of the paper discussed in the discussion section. An exception would be IMHO if the immobilisation procedure is well established and proven to result in fully active enzyme, than this would be mentioned in the background part of the introduction, with appropriate references. Of course, you'd still have to very that your preparation is fully active, and this should be mentioned under results.

Kindly see this reference

Procedia Engineering Dec 2013 68

DOI 10.1016/1 prow got 2013.12.200

It will be useful

Perhaps you could explore your system in other gold electrode (commercial for exemple). I`m not an explanation yet for this fact, I`m sorry Zahra.

James E Hanson I always use the same wavelength on ellipsometry and is not variable during the measurement. Thank so much for your time it already helped a lot!

There are a number of suppliers . Check google search .

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

For L-B-L technique you need strong binder and pH control, maybe you try with chitosan. You can also try screen printing or dip coating method with TiO2 paste. Ref:

Fabrication of screen-printing pastes from TiO2 powders for dye-sensitised solar cells

Look at the normal ELISA procedure and work on those buffers accordingly !!

Add 3-4% solution of sodium alginate prepared in 0.1 M phosphate (sodium) buffer (pH 7) by warming at 50 deg C. Allow to cool to room temperature, then add 1ml of your sterile enzyme stock solution earlier mixed with 9 ml of sodium alginate solution. Suspend drop wise into pre-chilled calcium chloride solution of concentrations say 0.1, 0.2, 0.3, 0.4 or 0.5 M. Gently stir mixture at 4°C for 2 h. Recovered beads formed by filtration and wash with distilled water. Store beads in 0.1 M phosphate (sodium) buffer (pH 7.0) at 4°C.

Different crops can grown in different soil. Some thrive better when stressed. That said, in general the balancing of Carbon and beneficial soil microbes to achieve an ideal Carbon to Nitrogen ratio of 24 : 1 has been generally considered good. This increases the organic matter and creates an environment of high productivity. I would though focus on whatever crop you are studying and different plans thrive in different environments and stress.

Additionally, I also found this link that summarized some discussion of C:N ratio and its impact on soil.

https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/carbon-to-nitrogen-ratio

Thats a difficult ask Saurabh....if you are talking of plant nitrogen , its plant physiology and perenniality that decide , how much n is to be mobilized and how much to be redistributed towards active sink sites....

Yes, you can utilize a crude enzyme extract for immobilization. I complete agree with Carlos Vera, you can go ahead and prepare CLEAs for the enzyme of your interest.

Please find the recent paper from my research group.

Waiting for your response

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.

Alternatively, you can use Microfluidics systems to immobilize the whole larva for CNS imaging, while the larva is fully intact.

Hi Yaaser Q Almulaiky,

According to the question, I would like to place my point of view regarding it:

The immobilized enzyme activity generally depends upon the type of support (immobilization medium) it has. When an enzyme is immobilized, then its microenvironment conditions gets changed in terms of conformational change in enzyme structure. Consequently, there will be change in its denaturation activation energy (Ead), I.e. Ead will be increased which makes enzyme more resistant to different thermal conditions. If we analyze it in a subtle way, As enzymes has different forms, some are dimer or polymeric as well in respective to their subunits. (There must be immobilization of enzyme properly in accordance to its subunits, otherwise it may fluctuate the optimum stability condition). The immobilized enzyme subunits acts as monomers (doesn't dissociates), nevertheless shows good stability with the immobilizer. Similarly, pH conditions works. Example, FTase (fructosyltransferase) enzyme shows good stability under pH 6 from its general stability level (pH 4.5). Similar kinds of points have been evaluated to justify the stability and working mechanisms according to the methodology and some calculations.

I hope this will help you to clear some of your doubts.

Thank you,

Regards.

Boronic acid might be immobilized onto cellulose paper via cross-linking which is an irreversible method done by the creation of intermolecular cross- linkages between the molecules of the enzyme by covalent bonds.

obrigado!

Hello Piravin Raj Barthasarathy

-20ºC is the recommended temperature when the GOx enzyme is under a solid and dry form especially when you are considering long term storage (see paper attached).

Performing the immobilization of GOx at room temperature for 24 hours could affect its activity but, as Siba Moussa mentioned, glutaraldehyde could help its stabilisation.

Working with GOx and biosensors, I recommend a storage (immobilisation process) at 4ºC especially because the GOx is dropcast within a solution.

I hope this helps.

Best regards,

Marie B.

We can use an overall effectiveness factor to help us analyze diffusion, flow, and reaction in packed beds especially considering a situation where external and internal resistance to mass transfer to and within the pellet are of the same order of magnitude. We can define an overall effectiveness factor that is based on the bulk concentration.

Look carefully at your data. since You list quantum yield, I assume the color is fluorescence. Was the original fluorescence green or a combination of blue and yellow - but now the yellow is gone? If it is a shift in the color from green to blue (higher energy, greater difference between the excited and ground states), either the ground state is stabilized or the excited state destabilized.

Thank you very much!! I am new in doing it so, I realize it is necessary to review some literature. :)

A primary amine can be used to immobilize an oligo to a surface that is derivatized with carboxylic acid groups using a carbodiimide such as EDC to form an amide bond between them.

Dear Hyou-Arm Joung

I hope the following is helpful:

First of all I would make the calibration for Lowry or Bradford using cellulase itself, not BSA. The slopes may differ a lot for different proteins. Secondly, the enzyme is diluted strongly in the washings, which may be a source of error, too. Consider to concentrate the enzyme in the washings using ultrafiltration membrane, to the volume of starting reaction mixture and then measure both protein concentration and enzyme activity. Finally make sure that no glutaraldehyde or tannin is leaking from the carrier. These may affect protein estimation, at least with Bradford.

Dear, Initially couple appropriate linker to carboxylic group and attach amino group containing nanoparticles. Carbodiimide can activate carboxylic group.

Thank you, Farrukh!