色氨酸与功能单体自组装体系的分子间非共价相互作用

利用荧光猝灭分析、 三维荧光光谱和计算机模拟等方法分别研究色氨酸(L-Trp)与己二酸二酰肼(ADH)、 双丙酮丙烯酰胺(DAAM)和丙烯酸(AA)3种功能单体间的非共价相互作用. 考察了荧光猝灭机理、 色氨酸-功能单体的相互作用强弱以及作用力类型. 研究结果表明, 功能单体对L-Trp的荧光猝灭过程是由于形成不发射荧光的复合物而引起的静态猝灭; L-Trp与功能单体的结合常数较大, 所形成的复合物较稳定, 非共价作用中以氢键和静电作用贡献最大; L-Trp主要通过羧基与功能单体产生相互作用, 芳杂环与功能单体间的相互作用相对较弱; L-Trp与功能单体相互作用的强弱顺序为L-Trp-ADH > L-Trp-DAAM > L-Trp-AA.     

Transfer and distribution of L-tryptophan between W/O and O/W, and between W/O and bicontinuous microemulsions through the study of interphase electric properties

The influence of L-tryptophan (L-Trp) on the interphase electric properties of sodium dodecyl sulfate (SDS)/n-pentanol (n-C(5)H(11)OH)/water (H(2)O) system was studied by the method of AC impedance, and the distribution of L-Trp between the microemulsion phases with different structures in SDS/n-C(5)H(11)OH/H(2)O system was determined by UV-vis spectroscopy. The results show that L-Trp transfers from the lower phase (O/W or bicontinuous) to the upper phase (W/O), and that a small amount of SDS transfers from the upper phase to the lower phase correspondingly with the increase of the total SDS content at a constant weight ratio of n-C(5)H(11)OH/H(2)O=50/50 and a total L-Trp concentration of 1.0 x 10(-4) mol/L. In addition, the concentrations of L-Trp in both the upper and lower phases increase, but the SDS content decreases slightly in the upper phase and increases appreciably in the lower phase with the increase of the total L-Trp concentration at a constant total content of SDS. The interphase capacitance and the interphase charge-transfer current of the system increase, but the interphase resistivity decreases.     

基于表面活性剂增敏有序介孔SiO2修饰碳糊电极测定L-色氨酸

利用水热法合成得到有序介孔二氧化硅( OMS),采用X射线衍射、N2吸附-脱附、透射电镜等技术对其进行了表征。制备OMS修饰碳糊电极( OMS/CPE),采用循环伏安和交流阻抗技术考察了修饰电极的电化学性能,发现与裸碳糊电极(CPE)相比,OMS/CPE具有更大的电活性面积和更快的电子传导速率。研究了L-色氨酸(L-Trp)在表面活性剂十二烷基磺酸钠(SDS)存在下,在OMS/CPE上的电化学行为和动力学性质,结果表明,OMS和SDS具有良好的协同作用,能明显增加L-Trp的响应信号; SDS存在下,L-Trp在修饰电极上的电化学氧化是2电子和2质子参加的不可逆过程,电极过程受吸附控制。优化了表面活性剂浓度、富集时间、富集电位、介质 pH 值等测定参数,在最优条件下, L-Trp 氧化峰电流与其浓度在8.0×10-8~4.0×10-6 mol/L范围内呈良好线性关系,检出限为7.0×10-8 mol/L(S/N=3)。本方法用于氨基酸口服液样品的加标回收检测,回收率为99.6％~102.6％。     

L-色氨酸分子印迹传感器敏感膜的制备与性能

以L-色氨酸(L-Trp)为模板分子, 邻苯二胺(o-PD)为功能单体, 在金电极表面原位合成了分子印迹聚合物敏感膜; 通过循环伏安法(CV)、 差示脉冲伏安法(DPV)和电化学阻抗谱法(EIS)考察了该电极的性能. DPV测试结果表明, 在1×10^-8~2×10^-7 mol/L范围内, 峰电流与L-Trp的浓度呈线性关系, 检出限为0.3×10^-8 mol/L; 选择识别性实验结果表明, L-Trp印迹敏感膜的印迹因子达到3.72, 相对于干扰物的选择因子均大于1, 对与L-Trp结构相似的L-酪氨酸(L-Tyr)的选择因子也达到2.30, 说明该印迹膜对L-Trp具有良好的选择性; 识别过程动力学研究结果表明, 印迹膜对L-Trp的识别是一个两步连续发生的过程, 即快结合过程和慢吸附过程.     

分子印迹聚吡咯/Fe3O4复合材料的制备及其在识别色氨酸光学异构体中的应用

首先利用共沉淀法合成了磁性材料Fe3O4,再以吡咯为单体,L-色氨酸为模板分子,采用化学聚合法使吡咯在Fe3O4表面发生原位聚合,同时通过分子间的作用力以及氢键作用将模板分子掺杂到Fe3O4表面的聚吡咯中,从而制备了分子印迹聚吡咯/Fe3O4复合材料,并且利用该材料的磁性质实现固液分离。在1 mol/L的NaOH溶液中,通过施加1 V的电位使聚吡咯发生过氧化从而使L-色氨酸模板分子脱掺杂。根据分子印迹的原理,该分子印迹的复合材料可用于识别L-色氨酸光学异构体。将扫描电镜、X射线衍射及电化学法用于该分子印迹复合材料的表征。将该材料填入到多孔陶瓷管,将L-和D-色氨酸溶液分别流过该多孔陶瓷管,流出液用高效液相色谱检测,发现该复合材料对于L-色氨酸的富集能力接近D-色氨酸的两倍,说明该复合材料具有作为手性识别色谱固定相的潜力。     

全二维微柱液相色谱-质谱鉴定人胃癌组织中的差异蛋白质

构建了以阳离子交换色谱-反相色谱(SCX-RPLC)为分离模式的新型全二维微柱液相色谱-质谱分离平台.采用了醋酸铵缓冲液梯度洗脱,实现了第一维肽段的分步洗脱,洗脱的肽段经富集除盐后通过接口进入反相色谱微柱,通过线性梯度实现第二维进一步分离,最后进入质谱进行检测.采用此平台分析了人胃癌组织与正常组织提取蛋白质信息,其中正常胃组织鉴定蛋白质数为537个,而癌症组织鉴定蛋白质数目为506个.对胃癌和正常组织两种提取蛋白质酶解产物的蛋白质检索结果进行比较分析,将鉴定的蛋白质按照物理性质进行分布,找出正常组织与癌症组织间蛋白质差异,筛选出一种可能发生变异的癌症特有蛋白.     

Application of cation-exchange solid-phase extraction for the analysis of amino alcohols from water and human plasma for verification of Chemical Weapons Convention

The analysis of nitrogen containing amino alcohols, which are the precursors and degradation products of nitrogen mustards and nerve agent VX, constitutes an important aspect for verifying the compliance to the CWC (Chemical Weapons Convention). This work devotes on the development of solid-phase extraction method using silica- and polymer-based SCX (strong cation-exchange) and MCX (mixed-mode strong cation-exchange) cartridges for N,N-dialkylaminoethane-2-ols and alkyl N,N-diethanolamines, from water. The extracted analytes were analyzed by GC-MS (gas chromatography-mass spectrometry) in the full scan and selected ion monitoring modes. The extraction efficiencies of SCX and MCX cartridges were compared, and results revealed that SCX performed better. Extraction parameters, such as loading capacity, extraction solvent, its volume, and washing solvent were optimized. Best recoveries were obtained using 2 mL methanol containing 10% NH(4)OH and limits of detection could be achieved up to 5 x 10(-3) microg mL(-1) in the selected ion monitoring mode and 0.01 microg mL(-1) in full scan mode. The method was successfully employed for the detection and identification of amino alcohol present in water sample sent by Organization for Prohibition of Chemical Weapons (OPCW) in the official proficiency tests. The method was also applied to extract the analytes from human plasma. The SCX cartridge showed good recoveries of amino alcohols from human plasma after protein precipitation.     

Dual-purpose sample trap for on-line strong cation-exchange chromatography/reversed-phase liquid chromatography/tandem mass spectrometry for shotgun proteomics. Application to the human Jurkat T-cell proteome

A dual-purpose sample-trapping column is introduced for the capacity enhancement of proteome analysis in on-line two-dimensional nanoflow liquid chromatography (strong cation-exchange chromatography followed by reversed-phase liquid chromatography) and tandem mass spectrometry. A home-made dual trap is prepared by sequentially packing C18 reversed-phase (RP) particles and SCX resin in a silica capillary tubing (1.5 cm x 200 microm I.D. for SCX, 0.7 cm x 200 microm for RP) ended with a home-made frit and is connected to a nanoflow column having a pulled tip treated with an end frit. Without having a separate fraction collection and concentration process, digested peptide mixtures were loaded directly in the SCX part of the dual trap, and the SCX separation of peptides was performed with a salt step elution initiated by injecting only 8 microL of NH4HCO3 solution from the autosampler to the dual trap. The fractionated peptides at each salt step were directly transferred to the RP trap packed right next to the SCX part for desalting, and a nanoflow LC-MS-MS run was followed. During the sample loading-SCX fractionation-desalting, flow direction was set to bypass the analytical column to prevent contamination. The entire 2D-LC separation and MS-MS analysis were automated. Evaluation of the technique was made with an injection of 15 microg peptide mixtures from human Jurkat T-cell proteome, and the total seven salt step cycles followed by each RPLC run resulted in an identification of 681 proteins.     

Electrocatalytic oxidation of L-tryptophan using copper hexacyanoferrate film modified gold nanoparticle graphite-wax electrode

A novel copper hexacyanoferrate (CuHCF) film modification on cysteamine (Cys)-gold nanoparticle (AuNp) graphite-wax (GW) composite electrode was achieved for the quantitative determination of L-Tryptophan (L-Trp) at a reduced overpotential of 400mV in comparison with the bare Cys-AuNp-GW composite electrode. This modified electrode exhibited a well resolved pair of redox peaks corresponding to the hexacyanoferrate (II/III) reactions of CuHCF film at a formal potential of 0.65 V at a scan rate of 20 mV s(-1). Electrochemical impedance spectroscopy (EIS) studies with the modified electrode showed a very low charge transfer resistance to the electron transfer kinetics of Fe(II)/Fe(III) reactions. A linear range of 8.5×10(-7) M to 1.2×10(-4) M with a detection limit of 1.85×10(-8) M was achieved for the determination of L-Trp with a sensitivity of 0.1198 μA/μM. The influence of ultrasonication on the stability of the CuHCF film modified electrode was investigated. In addition, the CuHCF film modified electrode displayed an excellent reproducibility towards the real time analysis of L-Trp in commercial milk samples.     

Spectroscopic studies on the inclusion interaction of p-sulfonatocalix[6]arene with vitamin B6

The formation of the inclusion complex of p-sulfonatocalix[6]arene (SCX6) with different forms of vitamin B₆ (VB₆) was studied by using fluorescence spectroscopy. VB₆ can exist in one of three forms (the acidic form, neutral zwitterionic form and basic form) depending on pH. The fluorescence intensities of acidic and basic forms of VB₆ remarkably decreased in presence of SCX6. SCX6 preferred to form 1:1 inclusion complexes with acidic and basic forms of VB₆ but hardly form inclusion complex with neutral zwitterionic form. According to the nonlinear curve fitting method, the inclusion constant (K) for the formation of inclusion complexes of acidic and basic forms of VB₆ with SCX6 were evaluated to be 1.4?×?10⁴ and 9?×?10³ L/mol, respectively. The binding affinity of SCX6 towards acidic form is attributed to hydrogen bonds and hydrophobic interaction, furthermore, additional electrostatic interaction also plays a crucial role. The possible inclusion mode was given by ¹H NMR technique.     

Liquid chromatographic analysis of glucosamine in commercial dietary supplements using indirect fluorescence detection

A method of using indirect fluorescence detection is evaluated for the analysis of glucosamine in commercial dietary supplements following chromatographic separation. In this method, the eluting analyte, glucosamine, was detected by monitoring an increase in the fluorescence signal for L-tryptophan (L-Trp) or DL-5-methoxytryptophan (5-MTP) after glucosamine complexed with a copper(II) ion and released either L-Trp or 5-MTP from a copper(II) complex, which is introduced postcolumn. The fluorescence of L-Trp and 5-MTP are quenched when complexed with the copper(II) ion. The results obtained using indirect fluorescence detection are compared with the results obtained for precolumn derivatization with phenylisothiocyanate. Statistical analysis is performed to compare the results obtained for the two postcolumn interaction components, Cu(L-Trp)2 and Cu(5-MTP)2, as well as the results obtained using the indirect fluorescence detection method and a precolumn derivatization method. The indirect fluorescence detection method provided an alternative to precolumn derivatization for determining the concentration of glucosamine in commercial dietary supplements. The stability of the glucosamine-o-phthalaldehyde-3-mercaptopropionic acid derivative is also evaluated to investigate the applicability of the popular precolumn derivatization reagent, o-phthalaldehyde-3-mercaptopropionic acid, for this analysis.     

The mechanism of substrate inhibition in human indoleamine 2,3-dioxygenase

Indoleamine 2,3-dioxygenase catalyzes the O(2)-dependent oxidation of L-tryptophan (L-Trp) to N-formylkynurenine (NFK) as part of the kynurenine pathway. Inhibition of enzyme activity at high L-Trp concentrations was first noted more than 30 years ago, but the mechanism of inhibition has not been established. Using a combination of kinetic and reduction potential measurements, we present evidence showing that inhibition of enzyme activity in human indoleamine 2,3-dioxygenase (hIDO) and a number of site-directed variants during turnover with L-tryptophan (L-Trp) can be accounted for by the sequential, ordered binding of O(2) and L-Trp. Analysis of the data shows that at low concentrations of L-Trp, O(2) binds first followed by the binding of L-Trp; at higher concentrations of L-Trp, the order of binding is reversed. In addition, we show that the heme reduction potential (E(m)(0)) has a regulatory role in controlling the overall rate of catalysis (and hence the extent of inhibition) because there is a quantifiable correlation between E(m)(0) (that increases in the presence of L-Trp) and the rate constant for O(2) binding. This means that the initial formation of ferric superoxide (Fe(3+)-O(2)(?-)) from Fe(2+)-O(2) becomes thermodynamically less favorable as substrate binds, and we propose that it is the slowing down of this oxidation step at higher concentrations of substrate that is the origin of the inhibition. In contrast, we show that regeneration of the ferrous enzyme (and formation of NFK) in the final step of the mechanism, which formally requires reduction of the heme, is facilitated by the higher reduction potential in the substrate-bound enzyme and the two constants (k(cat) and E(m)(0)) are shown also to be correlated. Thus, the overall catalytic activity is balanced between the equal and opposite dependencies of the initial and final steps of the mechanism on the heme reduction potential. This tuning of the reduction potential provides a simple mechanism for regulation of the reactivity, which may be used more widely across this family of enzymes.     

Substrate recognition and selection by the initiation module PheATE of gramicidin S synthetase.

The initiation module of non-ribosomal peptide synthetases (NRPS) selects and activates the first amino acid and serves as the aminoacyl donor in the first peptide bond-forming step of the NRPS assembly line. The gramicidin S synthetase initiation module (PheATE) is a three-domain subunit, recognizing L-phenylalanine (L-Phe) and activating it (by adenylation domain) as tightly bound L-phenylalanyl-adenosine-5'-monophosphate diester (L-Phe-AMP), transferring it to the HS-phosphopantetheine arm of the holo-thiolation (holo-T) domain, and then epimerizing it (by epimerization domain) to the D-Phe-S-4'-Ppant-acyl enzyme. In this study, we have assayed the selectivity of the PheATE adenylation domain with a number of proteinogenic amino acids and observed that three additional amino acids, L-Tyr, L-Trp, and L-Leu, were activated to the aminoacyl-AMPs and transferred to the HS-phosphopantetheine arm of the holo-T domain. Hydrolytic editing of noncognate aminoacyl-AMPs and/or aminoacyl-S-4'-Ppant-acyl enzymes by the enzyme was not observed by three different assays for adenylation domain function. The microscopic reaction rates and thermodynamic equilibrium constants obtained from single-turnover studies of reactions of L-Phe, L-Trp, L-Tyr, and L-Leu with holoPheATE allowed us to construct free energy profiles for the reactions, revealing the kinetic and thermodynamic basis for substrate recognition and selection. In particular, the rates of epimerization of the L-aminoacyl-S-enzyme to the D-aminoacyl-S-enzyme intermediate showed reductions of 245-, 300-, and 540-fold for L-Trp, L-Tyr, and L-Leu respectively, suggesting that the epimerization domain is an important gatekeeper for generation of the D-Phe-S-enzyme that starts gramicidin S chain growth.     

Encapsulation of Metronidazole in Biocompatible Macrocycles and Structural Characterization of Its Nano Spray-Dried Nanostructured Composite

Due to the great potential of biocompatible cucurbit[7]uril (CB7) and 4-sulfonatocalix[4]arene (SCX4) macrocycles in drug delivery, the confinement of the pharmaceutically important metronidazole as an ionizable model drug has been systematically studied in these cavitands. Absorption and fluorescence spectroscopic measurements gave 1.9 × 10⁵ M⁻¹ and 1.0 × 10⁴ M⁻¹ as the association constants of the protonated metronidazole inclusion in CB7 and SCX4, whereas the unprotonated guests had values more than one order of magnitude lower, respectively. The preferential binding of the protonated metronidazole resulted in 1.91 pH unit pK_a diminution upon encapsulation in CB7, but the complexation with SCX4 led to a pK_a decrease of only 0.82 pH unit. The produced protonated metronidazole–SCX4 complex induced nanoparticle formation with protonated chitosan by supramolecular crosslinking of the polysaccharide chains. The properties of the aqueous nanoparticle solutions and the micron-sized solid composite produced therefrom by nano spray drying were unraveled. The results of the present work may find application in the rational design of tailor-made self-assembled drug carrier systems.     

STUDIES ON THE CRYSTAL STRUCTURE OF Al-(L-TRYPTOPHAN) INSULIN AT 2.1RESOLUTION

In order to study the biological effect of alterations to the N-terminus of the insulin A-chain, we have determined the crystal structure of Al-(L-Trp) insulin and discovered that it belongs to the trigohal system with space group R3. The parameters oof the unit cell are a=b=80.3A, c=37.5A. The model was adjusted and refined by using a stereochemically-restrained least squares program, assisted by manual revision of the model based on the difference Fourier map, to a final R-factor of 0.195. The main and side chains of both Al-(L-Trp) residues in the asymmetric unit are well ordered. It was found that the Al-Trp residue of molecule I occupied two distinct positions. We have proposed from the results of the three-dimensional structure that the 4-zinc insulin hexameric form is a stored state of insulin molecules in a conformation of low activity. The structural details of the insulin molecule and its structure and function relationship have also been discussed.     

Spectroscopic studies of water-soluble sulfonated calix[6]arene

Fluorescence and absorption studies of water-soluble sulfonated calix[6]arene (SCX6) are reported. Water-soluble calixarenes are potentially useful as host molecules for luminophores, and studies of their spectroscopic characteristics are therefore crucial. The absorption and fluorescence spectra of these molecules in aqueous solution were collected, analyzed, and compared with 4-hydroxybenzene sulfonate at different pHs. A red shift in the absorption spectrum and a change in the fluorescence spectrum of the calixarenes are observed upon an increase in pH from 2.0 to 13.0. Some of these spectroscopic changes are attributed to intramolecular hydrogen bonding between adjacent hydroxyl groups of SCX6 after proton disassociation. The formation of excimers between phenolic groups in the calixarene molecule is proposed. In addition, inner-filter effects of SCX6 are discussed. These inner-filter effects prove to be a disadvantage for the use of SCX6 as a host molecule for complexation studies by use of fluorescence probes whose absorption spectra overlap with those of calixarenes.     

Polymethacrylate-type monoliths functionalized with chiral amino phosphonic acid-derived strong cation exchange moieties for enantioselective nonaqueous capillary electrochromatography and investigation of the chemical composition of the monolithic polymer

In situ prepared monolithic poly(glycidyl methacrylate-co-ethylene dimethacrylate) (poly(GMA-co-EDMA)) capillary columns were activated to reactive thiol-monoliths and subsequently functionalized with (S)-N-(4-allyloxy-3,5-dichlorobenzoyl)-2-amino-3,3-dimethylbutanephosphonic acid as chiral selector by radical addition to afford enantioselective strong cation exchanger (SCX) capillary columns (100 microm inner diameter (ID)). These monolithic capillaries were devised for the enantioseparation of chiral bases by nonaqueous and aqueous capillary electrochromatography (CEC) and the results obtained for mefloquine and its tert-butylcarbamate as test compounds were compared to those obtained with particulate silica-based analogs (packed columns). Despite abolishment of nonspecific ionic interactions between the cationic solutes and residual silanols that may diminish separation factors of the silica-based chiral SCX particles, the poly(GMA-co-EDMA)-supported SCX monolith did not, as expected, show better enantioselectivities, which was assumed to be due to detrimental nonspecific interactions between the analytes and the lipophilic polymer backbone. In order to minimize these unfavorable contributions, less lipophilic monoliths were developed by copolymerization of different amounts of the hydrophilic monomer 2-hydroxyethyl methacrylate (HEMA) with GMA and EDMA, leading to GMA-co-HEMA-co-EDMA-terpolymeric monoliths. By this increase of the hydrophilicity of the monolithic support the enantioselectivity of the resultant SCX stationary phase could be enhanced and reached values comparable to the packed silica-based enantioselective SCX capillaries. Additionally, the mobile phase composition and other variables were examined and it could be shown that the separation factors are considerably affected by diverse parameters such as acetonitrile-methanol ratio and type and concentration of the counterion. Mefloquine enantiomers could be separated with alpha-values up to 1.56 and a maximum plate count of ca. 60,000 m(-1) could be achieved.     

On-line parallel reversed phase two-dimensional liquid chromatography for high-throughput analysis of complex proteomic samples

A comprehensive two-dimensional liquid chromatographic system (2D SCX/RP) is con- structed with a 10-port-2-way valve using strong cation exchange chromatography (Hypersil SCX, 100 mm×4.6 mm I.D.) followed by reversed phase chromatography (Hypersil BDS C18, 15 mm×4.6 mm I.D.) to separate the complex peptides from globin peptic hydrolysate. After the sample was loaded on the SCX column, the phosphate buffer (pH 4.0) was used to elute the peptides. Then, elutes flowed through the interface and the peptides focused on the head of the trapping columns (Hypersil BDS C18, 15 mm×4.6 mm I.D.) but salt passed into the waste. After the valve was switched, the samples were flushed with a backward flow into the RP analytical column. The peptides on the SCX were eluted with 12 discontinuous steps linearly increasing salt concentrations. The peptides enriched on the trapping column were desalted and separated by the RP columns. The resolution and the resolved peaks of the 2D SCX/RP system were greatly increased and the total peak capacity reached as high as 2280.     

无孔单分散亲水性强阳离子交换固定相的制备及其在蛋白质快速分离中的应用研究

采用分散聚合法制备小颗粒种子及“一步种子溶胀聚合”法成功地制备了粒径为3.0 μm的无孔单分散亲水性交联聚甲基丙烯酸环氧丙酯树脂,其表面经水解、环氧化、再水解后与氯磺酸反应,制备了一种新型的强阳离子交换色谱填料（SCX）。详细考察了该填料对标准蛋白质的分离性能及流动相中盐的种类、有机溶剂、流速等对蛋白质保留的影响。实验结果表明,在流速为4 mL/min时,采用线性梯度洗脱,1.0 min内可快速分离4种标准蛋白质,蛋白质的保留符合阳离子交换色谱规律。将SCX应用于快速纯化鸡蛋清中的溶菌酶和猪心中的细胞色素-C,取得了较好的效果。     

Molecular‐Recognition‐Assisted pKa Shifts and Metal‐Ion‐Induced Fluorescence Regeneration in p‐Sulfonatocalix[6]arene‐Encapsulated Acridine

The host–guest interactions of cationic (AcH⁺) and neutral (Ac) forms of the dye acridine with the macrocyclic host p‐sulfonatocalix[6]arene (SCX6) were investigated by using ground‐state absorption, steady‐state and time‐resolved fluorescence, and NMR measurements. The cationic form undergoes significant complexation with SCX6 (K_eq=2.5×10⁴ M ^?1), causing a sharp decrease in the fluorescence intensity and severe quenching in the excited‐state lifetime of the dye. The strong binding of the AcH⁺ form of the dye with SCX6 is attributed to ion–ion interactions involving the sulfonato groups (SO₃^?) of SCX6 and the positively charged AcH⁺ at pH of approximately 4.3. Whereas, the neutral Ac form of the dye undergoes weak complexation with SCX6 (K_eq=0.9×10³ M ^?1) and the binding constant is lowered by one order of magnitude compared with that of the SCX6–AcH⁺ system. The strong affinity of SCX6 to the protonated form leads to a large upward pK_a shift (≈2 units) in the dye. In contrast, strong emission quenching upon SCX6 interaction and the regeneration of fluorescence intensity of the dye in the presence of Gd³⁺ through competitive binding have also been demonstrated.