Fluorescence quenching preliminary analysis between labeled concanavalin A and dendrimers for a glucose sensor - Fret, Pet and precipitation/aggregation mechanisms
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(2) [5] J. Sumner, N. Gralën, Eriksson-Quensel IB, "The molecular weights of canavalin, concanavalin A, and Concanavalin B". The Journal of Biological Chemistry 125 (1): 45–48 (September 1938. http://www.jbc.org/content/125/1/45. [6] H. Bittiger and H. Schnebli, Concanavalin A as a Tool, John Wiley & Sons, (1976). [7] R. J. Russell, M. V. Pishko, C. C. Gefrides, M. J. McShane, and G. L. Coté, “A fluorescence-based glucose biosensor using concanavalin A and dextran encapsulated in a poly(ethylene glycol) hydrogel,” Anal. Chem. 71(15), 3126–3132 (1999). [8] B. L. Ibey, H. T. Beier, R. M. Rounds, G. L. Coté, V. K. Yadavalli, and M. V. Pishko, “Competitive binding assay for glucose based on glycodendrimer-fluorophore conjugates,” Anal. Chem. 77(21), 7039–7046 (2005). http://www.ncbi.nlm.nih.gov/pubmed/16255607 [9] C. Huet, M. Lonchampt, M. Huet, and A. Bernadac, “Temperature effects on the concanavalin A molecule and on concanavalin A binding,” Biochim. Biophys. Acta 365(1), 28–39 (1974). http://www.ncbi.nlm.nih.gov/pubmed/4370427 [10] J. Kalb and A. Levitzki, “Metal-Binding Sites of Concanavalin A and their Role in the Binding of a-Methyl DGlucopyranoside” Biochem. J. 109, 669 (1968) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1186953/ [11] B. Ibey, H. Beier, R. Rounds, M. Pishko, and G. Coté, "Dendrimer based fluorescent glucose sensor for diabetic monitoring", Proc. SPIE 6094, 609401 (2006); doi:10.1117/12.645095 [12] R. Ballerstadt, C. Evans, R. McNichols, and A. Gowda, “Concanavalin A for in vivo glucose sensing: a biotoxicity review,” Biosens. Bioelectron. 22(2), 275–284 (2006). http://www.sciencedirect.com/science/article/pii/S0956566306000297 [13] J. R. Lakowicz, “Chapter 8: Fluorescence quenching” “Principles of Fluorescence Spectroscopy,” 3rd ed. Kluwer Academic/ Plenum, New York 2006). [14] K. E. Sapsford, L. Berti, and I. L. Medintz, “Materials for fluorescence resonance energy transfer analysis: beyond traditional donor-acceptor combinations,” Angew. Chem. Int. Ed. Engl. 45(28), 4562–4589 (2006). [15] Q. H. Xu, B. S. Gaylord, S. Wang, G. C. Bazan, D. Moses, and A. J. Heeger, “Time-resolved energy transfer in DNA sequence detection using water-soluble conjugated polymers: the role of electrostatic and hydrophobic interactions,” Proc. Natl. Acad. Sci. U.S.A. 101(32), 11634–11639 (2004). [16] R. J. Russell, M. V. Pishko, C. C. Gefrides, M. J. McShane, and G. L. Coté, “A fluorescence-based glucose biosensor using concanavalin A and dextran encapsulated in a poly(ethylene glycol) hydrogel,” Anal. Chem. 71(15), 3126–3132 (1999) http://pubs.acs.org/doi/abs/10.1021/ac990060r [17] M. J. McShane, Dennis P. O'Neal, Ryan J. Russell, Michael V. Pishko and Gerard L. Cote, "Progress toward implantable fluorescence-based sensors for monitoring glucose levels in interstitial fluid", Proc. SPIE 3923, 78 (2000); doi:10.1117/12.387127 [18] B. Ibey, H. Beier, R. Rounds, M. Pishko, and G. Coté, “Dendrimer based fluorescent glucose sensor for diabetic monitoring,” Progress in Biomedical Optics and Imaging - Proceedings of SPIE, (2006). [19] B. L. Ibey, H. T. Beier, R. M. Rounds, G. L. Coté, V. K. Yadavalli, and M. V. Pishko, “Competitive binding assay for glucose based on glycodendrimer-fluorophore conjugates,” Anal. Chem. 77(21), 7039–7046 (2005). [20] S. Mangold and M.Cloninger, “Binding of monomeric and dimeric Concanavalin A to mannose-functionalized dendrimers”. [21] D. L. Meadows and J.S. Schultz JS, “Fiber-optic biosensors based on fluorescence energy transfer,” Talanta, 35(2), 145-150, (1988). [22] Mo, W Antonín Vlcek Coord, “Highlights of the spectroscopy, photochemistry and electrochemistry of [M(CO)4( diimine)] complexes, M=Cr”, Chem. Rev. 230 (2002) 225-242..
(3) [23] Life technologies, Fluorescence SpectraViewer, http://www.invitrogen.com/site/us/en/home/support/ResearchTools/Fluorescence-SpectraViewer.html [24] Shima, S. and Sakai H,"Polylysine produced by Streptomyces". Agricultural and Biological Chemistry 41: (1977) 1807–1809.. 1. INTRODUCTION Diabetes is a disease that affects the production of insulin in a person’s body [1]. Although there are different kinds of diabetes, the main problem is based on the fact that the body cannot control its glucose level and this can cause imbalance in many of its processes. A high sugar level leads to severe damage to organs in the human body [2]. The main approach to measure glucose concentration in the body is the finger-stick method; however, this approach is inconvenient and uncomfortable for most users; it could be an easy way to get a disease if there is no proper care [3]. In order to solve this problem, a new fluorescence-based sensor, which is implanted under the skin, is being developed [4]. In this sensor, the glucose level is detected as a change in intensity proportional to blood glucose by making use of two components: labeled Concanavalin A, a lectin usually with four binding sites extracted from the jack-beans that can bind specifically to glycoproteins, sugar [5], and Dendrimers. Due to its properties, the binding sites of ConA are receptors for some saccharide. Tetrameric Con A has four binding sites and each one has the same chemical and physical configurations as the others [6]. Depending on the pH its structure can change becoming divalent at low values (below 5.5) and tetravalent at higher values (above 5.5 around the isoelectric point of the protein) [7]. Con A has compatibility and specificity to carbohydrate moieties found in glycoproteins, glycolipids and various sugars; therefore, current research is based on a competitive binding assay of fluorescently labeled ConA and a competing ligand: the dendrimer. Due to this last unity, the concentration of labeled ConA-dendrimer and labeled ConA-glucose will allow the monitoring of glucose changes by checking the fluorescence signal. The results have shown that when labeled ConA binds to the dendrimer in the absence of glucose the signal decreases to its minimum and it increases with the addition of glucose (as shown in figure 1) [3]. Both ConA and dendrimer are biocompatible and the toxicity in the human body is negligible [8]. At the same time it has been found that the ability for ConA to bind to glucose requires ions such as calcium and manganese divalent cations [9]. These cofactors allow for the binding of the sugar residues [10]. [Labeled ConA] + [Glucose] [Labeled ConA]+ [Dendrimer]. [Labeled ConA/Glucose] [Labeled ConA/Dendrimer]. [. =[ [. =[. /. ]. ][. ]. /. ]. ][. ]. By increasing glucose, the concentration of the ConA/Glucose complex will increase and the concentration of the complex ConA/dendrimer will decrease having an effect in the fluorescence. It is important to note that the equilibrium will be defined by the chemical affinity among the components, electronic interactions, and others variables that are fundamental to the sensitivity and functionality of the sensor. The decrease of the fluorescence intensity due to dissipation of the fluorophore’s electronic energy as a non radiative energy emission (heat, vibration, etc.), is called fluorescence quenching [11]. A variety of molecular interactions can result in this process including exited-state reaction, molecular rearrangements, energy transfer, ground-state complex formation and so on [12]. In theory, two types of quenching are discussed [13]. The first result from collisional encounters between the fluorophore and quencher and it is called dynamic quenching; in this case, the quencher must diffuse to the fluorophore during the lifetime of the exited state. Upon contact, the fluorophore returns to the ground state without emission of a photon. The second one is called static quenching. In this process, a complex is formed between the fluorophore and quencher making it nonfluorescent. All these imply molecular contact between the components (2Å) and chargecharge interactions or steric shielding can be interacting as well..
(4) Glucose Dendrimer. Labeled ConA. Figure. 1 Representation of the binding equilibrium among Concanavalin A, dendrimers and Glucose Resonance energy transfer RET, which is a mechanism of quenching, is a through-space interaction that occurs over longer distances and it is known as a radiationless mechanism (30 Å) [14]. When a photon excites an electron of the fluorophore emitting energy, part of it excites the quencher which can be fluorescent or nonfluorescent. Therefore, the process reduces the final intensity that is efficiency-related (that is dependent on the inverse sixth power of the intermolecular separation [15]). If both components are fluorescent chromophores the process is called FRET. In this study, the dye of Concanavalin A excites the fluorophore attached to the dendrimer; this energy is absorbed and the second emission is lower according to the absorption spectrum of the acceptor that must overlap the fluorescence emission spectrum of the donor (as shown in figure 2). The ConA-dendrimer complex has to satisfy the distance dependence of RET, so when glucose is added that distance between quencher and fluorophore is not allowed. It is important that the quenching that results of the binding ConA-dendrimer has showed no need labeled dendrimers so it gives arguments to say that other processes are happening besides FRET; despite the initial studies were found by this, the new dendrimers have showed FRET does not work as well as thought . Significant work has been done to find the appropriate ligand to improve the affinity between ConA and dendrimer. Initially, the investigation was focused on using dextran, a branched linear polysaccharide, to make a multivalent ligand with dyes to promote FRET [16]. The experiments showed promising results, but a small overall response to changes in glucose was observed [17]; then, the ligands were designed using different sugars residues as functional groups. A variety of them were made with various sizes, functional groups and volume, amongst other varying properties. Unlike the dextran approach, this new ligand known as dendrimers, have a high degree of molecular uniformity, narrow molecular weight distribution, specific size and shape characteristics, and a series of repetitive steps starting with a central initiator core [18]. Recent studies have worked with a new generation of dendrimers which are formed by mannose or glucose and amino groups on the surface; so, when ConA and dendrimer bind together, even in the absence of a donor/acceptor hetero-FRET system, the fluorescence intensity decreases. For many research studies,the fourth generation (G4) Polyamidoamine (PAMAM) dendrimers (Sigma, St. Louis, MO) has been used, with a theoretical molecular weight of 14,215 Da, a diameter of 45 Å and 64 terminal amine groups through the Michael addition of glucosyloxyethyl methacrylate. This glycosylated dendrimer shows potential for use as a competitive particle [19].Furthermore, work on the equilibrium constants has had significant progress in order to lead to consistent values of concentrations in the equilibrium; it was found that by adding sugar residues (methyl -mannosa) to the PAMAM dendrimer and changing the type of ConA (monomeric, dimeric or tetrameric form), the KCD magnitude can change easily [20]. This study makes use of dendrimers G2-glucose12-AMP12 (C393H684N144O96) with molecular weight of 8957.30 and G2glucose12-PPM12 (C393H672N132O108) with molecular weight of 8974.3604 which are different from PAMAM dendrimer because the spatial structure changes having a core with functional groups and branches. The first ones have twelve.
(5) Figure. 2 Donor and acceptor spectra for FRET. amino groups in its surface and twelve glucose residues as functional groups; the others have twelve hydroxyl groups in its structures and twelve glucose residues. Both are compared and studied to argue the proposed mechanisms. So far, the explanation as to why the fluorescence intensity changes in response to aggregation is due to FRET [21]. Light scattering was also proposed because of the size of the complex affecting the output signal [20]; however there are not strong studies about that. On other the hand encapsulation methods for the compounds has been a great topic of research and there are significant advances with hydrogels and polymeric meshes [3]. This research shows evidence that Photoinduced Electron Transfer (PET) is one of the main quenching mechanisms. In PET the electron donor and the electron acceptor form a complex. When light excites a molecule, an electron in a ground state orbital can be excited to a higher energy orbital, leaving a vacancy in a ground state orbital that can be filled by an electron donor. The electron in the high energy orbital can be donated to an electron acceptor. As a result a photoexcited molecule can act as a good oxidizing agent or a good reducing agent [22]. The amino groups belonging to the used dendrimers (or to the medium/ buffer) would be included as these quenchers. This paper will discuss how the different groups affect the fluorescence response Finally, the last mechanism that will be discussed is precipitation/aggregation. Dendrimers have a lot of binding sites that attach to ConA molecules; therefore, ConA-Dendrimer can become a big particle that can precipitate to the bottom of the container. According to this explanation there will be a concentration gradient depending on time and height that will have different responses. Assuming that the readings of the fluorescence intensity are always to the same spot, the signal is relative and it can become a problem for control. By adding glucose the bindings would change and precipitation could be reversible; evidently this process has an effect on the concentration of fluorophore in solution that would be detected by the fluorescence device. 2 .MATERIALS AND METHODS Labeled Concanavalin A Alexa Fluor 647 (type IV), dimethyl sulfoxide (DMSO), trizma- hydrochloride (Tris-HCl), Dglucose, Alexa Fluor 750, QSY 21- succinymidyl ester was purchased from Invitrogen (Carlsbad, CA), sodium bicarbonate (NaHCO3) and dextrose were purchased from Fisher Scientific (Waltham, MA). Hepes, sodium chloride, Distilled water used in experiments was collected from the Millipore filtration system at 18 M (Billerica, MA). Glycosylated labeled and unlabeled dendrimers averaging 12 glucose moieties and 12 amine or hydroxyl functional groups were received from Dr. Simanek’s lab group at Texas A&M University. In addition, 1 M hydrochloric acid (HCl), 1 M sodium hydroxide (NaOH), and 0.1 M phosphate buffered saline (PBS) was purchased from Sigma-Aldrich. Calcium chloride (CaCl2) was purchased from EMD Chemicals (Gibbstown, NJ). Sephadex beads (G75) were purchased from Amersham Biosciences (Waukesha, WI). Distilled water used in experiments was collected from the Millipore filtration system at 18 M (Billerica, MA). Tris buffer used in these experiments was created via the formulation of 0.15 M NaCl, 0.1 M Tris-HCl, 1 mM CaCl2, and 1 mM MnCl2, and titrated with the appropriate aliquots of 0.1 M NaOH & 0.1 M HCl to adjust the pH to 7.3. Poly-llysine, poly styrene sulfonate and polyethyleneimines were purchased from Invitrogen (Carlsbad, CA), bicarbonate was prepared under the same conditions of tris. 2.1 FRET: analyses among complexes The two types of G2 dendrimers were selected to do FRET analyses and to compare results. G2 Dendrimer type A has 12 amino groups and the second one has 12 hydroxide groups. 500 nM Succinyl-Concanavalin A (dimeric form) labeled with Alexa Fluor 488 was mixed with 250 nM Concanavalin A labeled with Alexa Fluor 647 into solutions of 500 nM dendrimer.
(6) NH2 type , 500 nM OH type and 500 nM G4 dendrimer in HEPES buffer at pH 7,4. The samples were analyzed by using the microplate reader with the following settings: excitation at 475 nm, emission from 505 nm to 705 nm, step 10 nm and 100 % of gain. In this way it was studied the bindings between the ConA-Dendrimers complexes in which the decreasing signal is related to FRET. It is supposed that Dendrimer is going to bind to many molecules of ConA regardless of whether it is Succinyl ConA Alexa fluor 488 or tetrameric ConA 647. After the scan, it was added glucose (500 mg/dL) to each sample. 2.2 FRET: analyses between Cona-dendrimer 500 nM Concanavalin A labeled with alexa fluor 647 was mixed into solutions of 500 nM G2 dendrimer (NH2) labeled with alexa fluor 750 and QSY 21. The samples were mixed and tested by using the microplate reader. After the scans, a glucose concentration of 500 mg/dL was added to the ConA-dendrimer solution. 2.3 Precipitation/ aggregation test Different solutions of Concanavalin A and G2 Dendrimer were prepared with the two different G2 Dendrimerl structures. Taking 1 mL of final volume 500 nM Labeled Concanavalin A with Alexa fluor 647 was mixed with 500 nM G2 glycodendrimer type A into TRIS buffer at 100 mg/dL and 1000mg/dL of glucose. At the same time, these experiments were repeated with 500 nM G2 glycodendrimer type B. After 10 minutes of waiting, the samples were centrifuged at 12K RPM for 5 minutes. Finally 200 uL of supernatant were taken and analyzed by using the microplate reader. The settings used were: excitation at 620 nm, emission from 650nm to 720nm, step 5 nm and 100 % of gain. 2.4 Addition of amino groups for PET Samples of 500 nM Labeled Concanavalin A with Alexa fluor 647 were mixed with Poly-L-Lysine (PLL) and 500 nM G2 dendrimer with amino groups into bicarbonate buffer at pH 10.25. Different concentrations of PLL were prepared from 0 % w/v to 0,01% w/v and the samples were analyzed by using the microplate reader at 620 nm of excitation, emission from 650nm to 720nm, step 5 nm and 100 % of gain. At the same time, varying solutions of 500 nM free dye (Alexa fluor 647) were prepared with PEI, Linear polyethyleneimines, from 0% ug/mL to 1000 ug/mL in HEPES buffer. This procedure was repeated with poly styrene sulfonate (PSS) as well. Making all these, the fluorophore was subjected to all amines groups to study PET process. PEI contains secondary amines and PSS tertiary amines. 2.5 Changing buffer The above experiments were prepared by using different buffers and pH in order to determine the effect of the molecules regarding PET and electronic interactions. HEPES, tris and bicarbonate buffer were used at medium and high pHs. 3. RESULTS AND DISCUSSIONS 3.1 FRET This experiment shows that the ConA-dendrimer complex could not be argued just by FRET and even when the behaviors of the graphs show promise, other processes may be playing a significant role. In the following experiments, when glucose is added, the fluorescence intensity for FRET is not coherent with the expected tendency. Dendrimer can bind to ConA independently if it is labeled with alexa fluor 647 or 488; however when both are together and have enough distance, FRET mechanism turns on. Proximity will be due to aggregation and bindings between them..
(7) Fluorescence Intensity (arb units). 55000. ConA-Dendrimer Complex. 45000. 488 ConA AF 448. 35000. ConA 647. 25000. G2 NH2. 15000. G2 OH. 5000. G4 NH2. -5000 505. 555. 605. 655. 705. Wavelength (nm). Fluorescence Intensity (arb units)). Figure 3. Fluorescence scans for the ConA-Dendrimer complex. Five samples were analyzed; there are two control responses that do not have dendrimer (ConA AF488 and ConA AF647). ConA-Dendrimer Complex. 3500 3000. ConA AF 448 488. 2500. ConA AF 647. 2000. G2 NH2. 1500 1000. G2 OH. 500. G4 NH2. 0 625. 645. 665 Wavelenght (nm). 685. 705. Fluorescence intensity (arb units). Figure 4. Fluorescence scans for the ConA-dendrimer complex in the range 625-705 nm. Five samples were analyzed; there are two control responses that do not have dendrimer (ConA AF488 and ConA AF647). 60000 50000 40000 30000 20000 10000 0. ConA-Dendrimer OH 0 Glucose 500 mg/dL Glucose 505. 555. 605. 655. 705. Wavelenght (nm) Figure 5. Fluorescence scans for the ConA-dendrimer OH complex at 0 and 500mg/dL of glucose The figures 3 and 4 show that the fluorescence intensity goes down for the fluorophore donor (488) and increases for the fluorophore acceptor (647) indicating that its emission could have been absorbed due to FRET as the theory makes mention. It is found that depending on the type of dendrimer the response can be higher and it is coherent due to the size of each one. For G4, there is more response due to its bigger size that involves more aggregation. However figure 5 shows that the signal is almost the same when glucose is added and it is inconsistent because it was expected have less quenching. Therefore it cannot be omitted that reabsorption of the output signal can be happening instead of FRET and.
(8) others processes have to be considered. In reabsorption, the transition of energy is due to radioactive mechanisms and it is possible even in long distances. In order to check better all these, the next experiment showed that FRET definitely is not coherent when glucose is added. Figure 6 shows that Alexa Fluor 647 and QSY21 can easily promote FRET, because the spectrum of the fluorescence emission for AF 647 can be overlaps the excitation spectra of the dendrimer QSY 21. The same spectrum for Alexa fluor 750 is less convenient for the same purpose; so it is expected the change of intensity will be higher for the first complex. However, the intensity responds resulted much sensitive for the second complex. In figure 8 is observed that when it was added glucose the increase of the peek for alexa fluor 647 is not proportional to the second peek (alexa fluor 750), this last keeps almost constant in the process.. Fluorescence Intensity (arb units). Figure 6. Emission spectrum for Alexa Fluor 647 (green) and excitation (absorption) spectrum for QSY 21 (blue) [23]. 500 nM AF 647 ConA & 500 nM G2 - QSY 21. 100000. 0 mg/dL 50000. 500 mg/dL. 0 650. 700. 750 Wavelength (nm). 800. Fluorescence Intensity (arb units). Figure 7. Fluorescence scans for the ConA-Dendrimer complex in the range 625-705 nm.. 500 nM AF 647 ConA & 500 nM G2 - Alexa Fluor 750. 40000 30000. 0 mg/dL. 20000. 500 mg/dL. 10000 0 650. 700. 750 Wavelength (nm). 800. Figure 8. Fluorescence scans for the ConA-Dendrimer complex in the range 625-705 nm..
(9) The graph 7 shows that the same behavior was found. The difference is that QSY 21 does not have an emission spectrum, so just one peek is seen. However the change is smaller than the dendrimer with AF 750 and this indicates that others mechanisms happen with a higher effect on the quenching when ConA- dendrimer is formed. 3.2 Precipitation. Fluorescence Intensity (arb units). It was observed blue-pellet at the bottom of centrifuge tube just for the sample that contained G2 dendrimer with amino groups; for the carboxyl, the solutions remained apparently the same. According to the figure 10 the NH2 supernatant indicates a significant change when glucose is present and there is a clear tendency to return to the initial value. Also as it was expected, the blue-pellet decreased at high glucose concentration. With respect to the OH dendrimer, the responses are not consistent and those differences could be just noise.. OH-Dendrimer. 160000 140000 120000 100000 80000 60000 40000 20000 0 650. 670 690 Wavelength (nm) No Dendrimer 0 100. 710 1000. Fluorescence Intensity (arb units). Figure 9. Fluorescence analysis for surfactant with G2 dendrimer-hydroxyl groups. The graph include four samples analyzed: two controls (free labeled ConA –no dendrimer and labeled ConA/dendrimer -0- ) and two concentration of glucose. NH2 Dendrimer. 140000 120000 100000 80000 60000 40000 20000 0 650. 670 690 Wavelength (nm) No Dendrimer 0. 710 100. 1000. Figure 10. Fluorescence analysis for surfactant with G2 dendrimer-amino groups. The graph include four samples analyzed: two controls (free labeled ConA –no dendrimer and labeled ConA/dendrimer -0- ) and two concentration of glucose Centrifugation just accelerated the process that would normally happen; so, this could argue that a gradient of fluorophore concentration is formed in Z axis and depending on where the fluorescent reading is , the respond can change. Time would be a new variable until it reaches the steady state. Precipitation test not only demonstrates that the ConA-dendrimer complex can become a large particle that can precipitate, it also shows that depending on chemical structure of the dendrimer the electronic interactions can be important. NH2 Dendrimer revealed high precipitation at lower volumes but with OH dendrimer no precipitation was observed; this could give a reason that the binding is much weaker in that complex..
(10) 3.3 Addition of amino groups It was found that amino groups from Poly-L-Lisine had a direct effect on the fluorescence respond when it was added into the solution of ConA and NH2 Dendrimer. According to the figure 11, the behavior at increasing PLL concentration seems to be the same when there is glucose presence at different concentrations. The reason that explains the analogy of responses could be related to the size of aggregation between ConA-dendrimer. As it was explained in the figure 1, the complexes are going to be aggregated and the amount of amino groups belonging to dendrimers will be greater and therefore increasing the concentration around the fluorophores. The precipitation test was employed in these samples as well and the blue-pellets at the bottom of the centrifuge tube were found. The pellets give evidence that the bindings are still happening even when a high concentration of amino groups is presented.. 500 nM Alexa Fluor 647 ConA in 10 mM Bicarbonate Buffer at pH 10.25. Fluorescence Intensity (arb units). 140000 120000 100000 80000 60000 40000 20000 0. 0 w/v% PLL 0.00001 w/v% PLL 0.0001 w/v% PLL 0.001 w/v% PLL 0.01 w/v% PLL. 650. 670. 690(nm) Wavelength. 710. Figure 11. Addition of amino groups into samples of ConA at different PLL concentrations.. 500 nM Alexa Fluor 647 ConA in 10 mM Bicarbonate Buffer at pH 10.25. Fluorescence Intensity @ 670 nm (arb units). 140000 120000 100000 80000 60000 40000 20000 0 0. 0,002. 0,004 0,006 [Poly-L-Lysine] (w/v%). 0,008. 0,01. Figure 12. Addition of amino groups into samples of ConA at different PLL concentrations.. +. Figure 13. NH2 and NH3 forms Dendrimers with amino groups in its structure have been more sensitive in glucose response than dendrimer with carboxylic groups and it is can be explained with PET. Amino groups belonging to them can be protonated depending on the pH; the NH2 form has one free electron pair in its last valence shell that can be easily donated when the electrons of the fluor-.
(11) ophore are excited. In this way, the electrons of amino groups (from the dendrimer or medium) can take those empty electron´s spaces preventing the return of the original exited electron. To evade PET mechanism the pH can be changed by favoring the NH3 + form (as shown in figure 13) but ConA structure could result influenced as well. According to the figure 11 and 12 the influence of amino groups is suitable to explain why the fluorescence can vary easily even if there is a little pH change. So, the buffer would be fundamental to control PET.. Fluorescence Intensity (arb. units). PLL is a synthetic amino acid chain that is positively charged. There are approximately 25-30 lysines attached to each other per chain – equivalent to 25-30 amine groups [24]. Figure 13 shows that the signal is been affected at increasing PLL concentration and the graph is similar when glucose is present; PEI (secondary amines) and PSS (tertiary amines) showed similar results (figures 14 and 15).. 500 nM ConA AF 647 in Tris. 70000 60000 50000 40000 30000 20000 10000 0. PLL: 0 ug/mL PLL: 1E-6 ug/mL PLL: 1E-5 ug/m PLL: 1E-4 ug/m PLL: 1E-3 ug/mL PLL: 0.01 ug/mL 650. 670 690 Wavelength (nm). 710. FLuorescence Intensity (arb units). Figure 14. Samples at different PLL concentrations. 100000. 500 nM free dye AF 647 in HEPES PSS: 0 ug/mL. 80000. PSS: 0,1 ug/mL. 60000. PSS: 1 ug/mL. 40000. PSS: 10 ug/mL. 20000. PSS: 100 ug/mL PSS: 1000 ug/mL. 0 650. 670 690 710 Wavelength ( nm). Figure 15. Samples at different PSS concentrations 3.4 Changing Buffer Dynamic quenching based on PET has been found by changing the medium; two ways have been implemented to prove it: by adding amines into the samples, and by changing the buffer. Tris, HEPES and Bicarbonate buffer presented different responses due to their chemical structures and buffer capability, it is apparent that depending on the chemical structure of the compound, the concentration of amine can either increase promoting PET in dynamic quenching or affect the chemical interactions. It was found that HEPES resulted more appropriate at neutral pHs and it is similar to the pH of the blood, bicarbonate buffer resulted more convenient due to charges (neutral) and simplicity of the molecule and tris appears to increase the signal sensitivity (amino groups are present). All the quenching that has been discussed center on the interaction ConA-dendrimer; the complex that is formed gives an idea that static quenching could involve whole the phenomenon. However it has been found that glucose presence in high concentrations does not have effect on the intensity and this never becomes as high as the original (when there are not quenchers); while it is known that ligand binding is determined by one equilibrium constant, at high concentration of glu-.
(12) cose the new complex ConA-glucose would tend to dominate (the equilibrium shifts into products). To answer this question there are two hypotheses that will have to be studied. The first includes all the mechanisms to dynamic quenching that are related to all diffusion processes between quencher and donor and the second one is based on electrical interactions that could be affecting the binding ConA-dendrimer; here the charges of the dendrimer would be the causes of the repulsion. Electrical interactions are definitely a concern because it would be another variable which would have effect on the equilibrium.. Figure 16. sensing chemistry. 4. CONCLUSION It was found that the amino groups belonging to dendrimer and medium are making possible PET mechanism and the fluorescence intensity is been strongly affected by adding these quenchers. Although it was added amines into the samples (dynamic quenching), the behavior for the ConA-dendrimer aggregation is expected to be analogous but more stable (static quenching). The aggregation size of ConA-Dendrimer would increase the amount of amines around the fluorophore that permit this mechanism. Also it was noticed that the different types of amines are affecting the fluorescent signal and the tendency seems to be exponential by increasing amine concentration. It would be good to run new experiments with dendrimer at different amount of amino groups. Precipitation test showed that the ConA-dendrimer complex can fall down making a gradient through the volume. This will be a real problem because the fluorescence intensity would be dependent on the time and position of lecture meaning that the signal can be affected to long-time. At the same time it was found that dendrimer OH does not precipitate and this could be due to electrical interactions; therefore, changing the chemical structure of the dendrimers might be the best way to monitoring glucose and OH groups has resulted as a new opportunity for studying because it will not permit PET and the electrical interactions will be lower (neutral group). Research is needed to explore the bindings between ConA- OH Dendrimer and the affinity among components. FRET is still a good way to work due to the design of the dendrimers were made to promote this mechanism and it is experimentally more viable. So, if it is possible to avoid the others mechanism by changing the functional groups or other properties, it will be possible to continue with the initial hypothesis. Otherwise PET mechanism can be used as an alternative approach but dendrimer structure has to be restructured. Definitively Precipitation, from this perspective, will become a problematic variable because the compounds would be encapsulated in microspheres or limited spaces. Light scattering and reabsorption of the emission by fluorophore need to be studied in detail and maybe more mechanism are associated which will have to be discovered to control in all aspect the sensor’s functionality.
(13) ACKNOWLEDGMENTS The author would like to thank Brian M. Cummins (Biomedical Engeneering Department, Texas A&M University, Texas, USA), doctoral student, for his help in developing this work, and the Optical Biosensing Laboratory for worthwhile discussions toward the realization of such a sensor..
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No obstante, como esta enfermedad afecta a cada persona de manera diferente, no todas las opciones de cuidado y tratamiento pueden ser apropiadas para cada individuo.. La forma
The expansionary monetary policy measures have had a negative impact on net interest margins both via the reduction in interest rates and –less powerfully- the flattening of the
