Drug Sensing Protein Crystals Doped with Luminescent Lanthanide

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Drug Sensing Protein Crystals Doped with Luminescent Lanthanide Complexes Guotao Sun, Jianguo Tang, Christopher D. Snow, Zhenhua Li, Yu Zhang, Yao Wang, and Laurence A. Belfiore Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.9b00642 • Publication Date (Web): 26 Aug 2019 Downloaded from pubs.acs.org on August 29, 2019

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Crystal Growth & Design

Drug Sensing Protein Crystals Doped with Luminescent Lanthanide Complexes Guotao Suna, Jianguo Tang*a, Christopher D. Snow*b, Zhenhua Lia, Yu Zhanga, Yao Wanga, Laurence A. Belfiore b a Institute

of Hybrid Materials, National Center of International Research for Hybrid Materials Technology, National Base of International Science & Technology Cooperation, College of Materials Science and Engineering, Qingdao University, Qingdao, 266071, P. R. China. Email: [email protected] b Department

of Chemical and Biological Engineering, Colorado State University, Fort Collins, Colorado 80523 USA. Email: [email protected] ABSTRACT: HEWL (hen egg white lysozyme) crystals are a model system for protein crystallization due to economical production and facile crystal growth. Here we grew small, rod-like HEWL crystals and proceeded with stepwise synthesis to dope the protein crystals with luminescent lanthanide complexes [(Eu(TTA)3phen] TTA = 4,4,4-trifluoro-1-(2-thienyl)1,3-butanedionato, phen = 1,10-phenanthroline). Several compounds (coumarin, tinidazole, and acridine orange) were observed to quench the lanthanide complexes in solution or embedded within crystals. Coumarin is a precursor for anticoagulant drugs and tinidazole is used to combat bacterial or protozoan infections. In contrast, acridine orange is a widely used, versatile fluorescent dye used to stain acidic vacuoles, RNA, and DNA in living cells. These results suggest that protein crystals may provide a feasible matrix for entrapping luminescent species with practical biosensing applications.

Introduction Recent years have seen growth in the engineering of nanoporous materials, including protein crystals, towards biological, catalysis, sensing and photonics applications.1-5 A key sub-discipline is the combination of the biomolecular recognition capabilities of proteins with the functional properties of non-biological components. For example, multiple research groups have focused on synthesizing gold nanoparticles within the solvent channels of protein crystals.6-9 Our research groups are also interested in synthesizing hybrid proteininorganic crystals.10-11 One important difference is that our non-biological components are luminescent rare earth complexes. In solution, lanthanide complexes (LCs) offer very useful photonics properties such as a narrow emission band, large Stokes shift and long luminescence lifetime.12-17 Nanoporous protein crystals may, in principle, serve as high capacity scaffolds to not only adsorb LCs nanoparticles, but also to organize the

luminescent groups in a three-dimensional lattice that is strikingly different than the organization within LC crystals or aggregates. Controlling the organization of luminescent groups in threedimensional materials is a frontier for luminescent materials. This work covers several technical and scientific aspects which are necessary for preparing proteinLC hybrid materials. First, we describe a protocol to produce abundant, small, high-aspect ratio, highquality protein crystals composed of the favorable model protein hen egg white lysozyme (HEWL). Tunable shape is useful for optimizing the crystals for downstream applications. For example, rodshaped crystals will have different guest molecule diffusion kinetics compared to crystals with a lower aspect ratio. Rod-like tetragonal HEWL crystals will have relatively long axial pores (along the c axis). Notably, the largest guest molecules capable of diffusing into tetragonal HEWL crystals are likely to be restricted to diffusion along the axial pores. Therefore, by tuning the crystal habit we might tune

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Crystal Growth & Design 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

the rate of guest molecule uptake or release. Thus, shape-tunable crystal growth is a goal for this study, via precise control of protein concentration, pH, salt concentration, PEG concentration, and temperature. A second key technical aspect of this project is stabilization of the host crystals. For most material science applications, protein crystals require a chemical cross-linking step to improve mechanical properties and tolerance to changes in the surrounding solution.18-20 Here we used glutaraldehyde, the most common reagent used to form covalent bonds between neighboring amino acids in HEWL crystals. We thus convert fragile non-covalent crystals into robust samples that retain apparent stability in many solutions including anhydrous ethanol.

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There are several advantages of using crystals with nanopores that are closely size matched to the guest molecules of interest. Hypothetically, such crystals can tightly constrain the orientation and spacing of guest LC. Additionally, size matched crystals can more easily trap guest complexes that must diffuse slowly, perhaps in single file (Figure 1). It is possible to imagine solvent pores that are so small that the guest molecule complexes can only diffuse when adopting specific conformations, orientations, or partial disassembly states in the cases of complexes. Finally, we demonstrate drug sensing applications for the resulting luminescent doped protein crystal materials. We show that tinidazole, coumarin, and acridine orange (AO) can quench the fluorescence of our model LC, [(Eu(TTA)3phen]. Coumarin is a natural plant product, and the key precursor chemical for widely used anticoagulant drugs.23-24 Tinidazole is an economical drug that is widely used for preventing anaerobic infections. For example, tinidazole is used to reduce postoperative infection after elective colorectal surgery and appendectomy.25 Acridine orange is a fluorophore that can distinguish DNA and RNA by giving a red or yellow fluorescence signal, and a potential model theranostic compound for tumors.26 We observed quenching effects for these compounds both in solution and inside LC-doped crystals. The resulting crystals are therefore conditionally fluorescent, depending on the presence of an analyte. This observation is the key prerequisite to justify further exploration of these fluorescent “protein pixel” materials for biosensing applications. The crosslinked protein crystal matrix is an interesting chassis from the biosensing perspective, since it is possible to produce crosslinked crystals that are not cytotoxic4, and permanent attachment of LC to the crystal interior might increase the biocompatibility of the LC. For drug-sensor and biosensor applications outside of living organisms, the presumed long-term biodegradability of the protein could also serve as an advantage.

A final key technical aspect of this work is the refinement of methods for synthesizing LC inside of pre-existing protein crystals rather than diffusing intact LC into the crystals or attempting to incorporate LC during crystal growth.11 Guest molecule transport is a challenging step because [(Eu(TTA)3phen] is prone to aggregation in a variety of solutions and we expect that [(Eu(TTA)3phen] aggregates (often >3nm in diameter) are too large to diffuse into solvent channels of tetragonal HEWL crystals (1-2.5nm according to J. C. Falkner ).21

Figure 1. Cutaway view of the HEWL crystal structure with the major axis running vertically.22 The cross-section plane is coloured by the distance to the nearest protein atom. For a size comparison, coumarin (cyan), acridine orange (yellow), LC (magenta), and tinidazole (green) are shown with a space-filling representation.

Materials and methods Reagents and solutions The following chemicals were obtained and used without further purification. Lyophilized hen egg white lysozyme (HEWL) was purchased from Hampton Research, Inc. 2-thenoylthrifluoroacetone

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(TTA, ≥98%) was purchased from Shanghai Macklin Biochemical Company. 1,10-phenanthroline (C12H8N2, Analytical Reagent (AR)), anhydrous ethanol (CH3CH2OH, AR), and europium(III) oxide (Eu2O3 , ≥ 99.99%) were purchased from Sinopharm Chemical Reagent Company. Sodium acetate tri-hydrate was purchased from BASF SE (Tianjin, P.R. China). Glutaraldehyde (≥50%) was purchased from Tianjin Bodi Chemical Company. Poly-(ethylene glycol) (PEG), MW 6000, was purchased from Admas Reagent Company. Acetic acid (AR) was purchased from Tianjin Hengxing Chemical Preparation Company.

Europium (III) chloride hexahydrate was dissolved in anhydrous ethanol. [(Eu(TTA)3phen] Crystals

assembly

inside

HEWL

A LC ligand solution was prepared by mixing equal volumes of 3 mM TTA and 1 mM phen in anhydrous ethanol solution and adjusting the pH to 7.4 with 5M NaOH. We adsorbed Eu3+ into crystals by soaking crosslinked crystals in a 1mM EuCl3 in anhydrous ethanol solution for 2 days. After the free EuCl3 supernatant solution was removed by centrifuge at 4000g for 3 mins, we adsorbed the ligand solution into the crystals for 1 hour. The resulting [(Eu(TTA)3phen]-doped crystals were washed with anhydrous ethanol and dried in a vacuum at 50C.

HEWL Crystal Growth Crystals were grown via the batch method using 1.5 mL centrifuge tubes. The crystallization buffer contained 0.5 M acetic acid, 16% NaCl, and 6% PEG 6000. After addition of all these reagents (acetic acid, NaCl, PEG), the pH of the crystallization buffer was adjusted to 3.2 using 5 M NaOH. HEWL was resuspended in a buffer containing 0.05 M sodium acetate tri-hydrate aqueous solution with the pH adjusted to 3.2 using glacial acetic acid. Then 300 microliters of 6 mg/mL HEWL was combined with 700 microliters of crystallization buffer in a centrifuge tube. The batch was rapidly sealed and incubated for 24 hours at 7C for crystal growth.

Quenching Luminescent Protein Crystals We used acridine orange (AO), tinidazole, and coumarin to quench LC in solution and inside luminescent protein crystals. First, a 100 mM AO, 100 mM coumarin solution, and a 0.1 mM [(Eu(TTA)3phen] solution were made, each in anhydrous ethanol. A 50 mM tinidazole solution was made in acetone because tinidazole is poorly soluble in anhydrous ethanol. We then combined solutions, adding AO, tinidazole, or coumarin to the [(Eu(TTA)3phen] solution, and adjusted the pH value to 7.4 by titrating with 5 M NaOH. For timeresolved luminescence measurements, protein crystals were placed into a Nest Petri dish (35mm), optimized for fluorescence microscopy. Crystal fluorescence emission intensity was quantified using a 20/30 PV™ microspectrophotometer (CRAIC) with an excitation wavelength of 365 nm.

Washing and Cross-Linking To maximize the accessibility of the HEWL crystal nanopores, we next removed free HEWL monomer by washing the crystals with crystallization buffer. The crystal growth batch was centrifuged at 2000g for 3 min, and the supernatant was removed. We repeated each centrifugation process 3 times. The final 1 mL replacement solution used to resuspend the crystals was the cross-linking solution (the crystallization buffer mixture which contained 4% glutaraldehyde). The 1 mL cross-linking solution was incubated at room temperature for 24 hr, at which point the crystals were visibly yellow. The batch of crosslinked crystals was centrifuged at 4000g for 3 mins and resuspended in ultrapure water.

Results and discussion It is important to control crystal size and aspect ratio because these factors can play a dominant role in guest molecule transport properties.27-29 For example, small crystals are advantageous for the diffusion of substrate and product molecules into catalytic crystals. Small crystals can more rapidly uptake and release guest molecules such as [(Eu(TTA)3phen]. Therefore, one goal for this study was to develop an in-house methodology to reliably

EuCl3 Synthesis in Solution A europium chloride hexahydrate solution was made by dissolving europium oxide in excess hydrochloric acid and stirring at 60 C for 10 hours. After drying in a vacuum at 40C for one day,



obtain high-quality HEWL crystals with varying aspect ratio (Supplementary Fig. S1). After testing multiple crystallization strategies (e.g. batch and sitting drop) and optimizing solution conditions (e.g. pH, temperature, salt concentration, and protein to precipitant ratio) (Supplementary Fig S2) we obtained a recipe of 0.5 M acetic acid (pH 3.2), 16% NaCl, and 6% PEG 6000. This condition led to the growth of small rod-like crystals (Fig. 2d-f).

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Figure 3. Time lapse imaging of [(Eu(TTA)3phen] synthesis inside a host protein crystal. The scale bars are 20 µm.

Figure 4. Laser scanning confocal microscopy (LSCM) z-stacks images of protein crystal that load [(Eu(TTA)3phen]. The 16 μm z-stack was taken in the middle of the crystal at 2 μm intervals. The scale bar is 40 μm.

Figure 2. Scanning electron microscopy images of (a) a crosslinked HEWL crystal with a balanced growth habit, (b) a crosslinked HEWL rod-like crystal grown in a crystallization buffer lacking PEG, (c) a crosslinked HEWL rod-like crystal grown at 15C (d) a crosslinked HEWL rod-like crystal grown at room temperature and (e) a crosslinked HEWL rod-like crystal loaded with [(Eu(TTA)3phen]. (f) Microspectrophometer image of a crosslinked HEWL crystal loaded with [(Eu(TTA)3phen]. The scale bars are 40 µm.

To assess the crosslinked HEWL crystals doped with [(Eu(TTA)3phen], we obtained the luminescence emission spectra for the crystals with a 20/30 PV™ microspectrophotometer (CRAIC) (Supplementary Fig. S3). We observed the characteristic large Stokes shifted emission band (~613 nm) for [(Eu(TTA)3phen] using a 365 nm ultraviolet excitation laser beam. We also observed the synthesis of [(Eu(TTA)3phen] inside protein crystal as a function of time (Fig. 3). As a control, we compared the fluorescence emission spectra for the doped crystal with the intrinsic fluorescence spectra of a [(Eu(TTA)3phen] solution (Supplementary Fig S4). As expected, the emission peaks were at same position. Notably, this loading timescale is comparable to the timescale that Cvetkovic and coworkers used to load the large dye molecule fluorescein into tetragonal HEWL crystals.34

To control crystal size, we modulated protein solubility. As expected, lower protein solubility resulted in smaller crystals.30-32 The connection between crystal aspect ratio and growth solution was less clear. Aspect ratio changes were likely caused by both salt concentration and pH. A pH change can modulate the surface charge of the HEWL, and a difference in surface charge can lead to differential growth rates between the {101} and {110} faces of tetragonal lysozyme crystals.21 Durbin and Judge et al. reported that decreasing pH value and lower salt concentration can increase HEWL aspect ratios, but the trend was non-linear.32-33

To determine whether the lanthanide complex was uniformly loaded throughout the interior of the protein crystals (as opposed to aggregated on the surface) we used laser scanning confocal microscopy (LSCM). Of the available confocal laser wavelengths, [(Eu(TTA)3phen] was best excited at 405 nm.11 We


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therefore used a 405 nm laser to observe the crystal interior via z-slices collected at 2 μm intervals (Fig. 4). In addition to the qualitative uniformity of the fluorescence images, there was quantitative uniformity. ImageJ analysis of the mean intensity inside and outside the crystal as a function of zposition suggested that the middle of the crystal was the brightest (Supplementary Fig. S5). This can be rationalized due to greater out-of-focus contributions to the focal plane when the focal plan is in the middle of the crystal. To further verify the existence of [(Eu(TTA)3phen] within the crystals, we used field emission scanning electron microscopy (FESEM) and high resolution transmission electron microscopy (HRTEM). Constituent elements of [(Eu(TTA)3phen] and protein crystals were detected (Fig. 5a-f). The elements F and Eu were found homogeneously spread throughout the protein crystals, further confirming that [(Eu(TTA)3phen] was uniformly distributed within protein crystals. The background colors of Fig. 5a-b were red or green because the protein crystals used for FESEM were fixed with a conducting resin composed of carbon and oxygen. Furthermore, we were able to observe nanocrystalline domains (Fig. 5g) with regular lattice fringes comparable to the lattice fringes we observed for [(Eu(TTA)3phen] that was crystallized in the absence of the protein crystals (Supplementary Fig S6). The presence of the LC in the crystal powder was further confirmed via EDX observation of Eu and F peaks (Fig. 5h).

Figure 5. FESEM images for a luminescent, LCdoped protein crystal. The elements from (a) to (f) were C, O, S, N, F, Eu. Characterization of [(Eu(TTA)3phen] recovered from ground, doped protein crystals via (g) HRTEM with a 2 nm scale bar and (h) EDX spectra collected for the area shown in panel a. For example, Hui Wei et al. reported gold nanoparticles with diameter more than 15 nm grown inside of HEWL crystals.6 That could be the case here, as the mean diameter of the domains observed in the HRTEM imagery was about 14 nm. However, we estimate the maximum diameter of a probe sphere that can fit within a solvent cavity in a nondistorted tetragonal HEWL crystal is 1.6 nm (Supplementary Figure S7).22, 35 The crystallinity of the observed nanoscale domains is not in doubt, since only nanocrystals could generate the observed lattice fringes. However, there are multiple possible explanations for how and when [(Eu(TTA)3phen] nanocrystals arise. First, crystal growth could occur within HEWL crystal imperfections, vacancies or other faults in the lattice. Second, the crystal growth could occur when the crystals are dried, a process that is likely to create additional lattice imperfections. Third, it may be possible that nanocrystal growth in the mother liquor could have a thermodynamic driving force sufficient to displace HEWL monomers from their lattice positions.

It is important to note that observation of nanoscale crystals in the HR-TEM experiment does not necessarily imply that this is the typical state for LC doped within the intact crystals. Growth of nonbiological particles inside of protein crystals in which the non-biological particle diameter grows beyond the pore diameter of the native crystal has been observed before.



Fourth, LC nanocrystals could be forming at the crystal surfaces rather than the crystal interior.

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To analyze coumarin luminescence quenching efficiency, we used a nonlinear Stern–Volmer (S–V) equation

Drug sensors represent one possible application for LC-doped crystals. To begin to explore this application direction, we sought to identify molecules that are relevant to human disease and can also modulate the fluorescence of doped crystals. We found that tinidazole, coumarin, and acridine orange (AO) had a distinct quenching effect on the [(Eu(TTA)3phen] luminescence, even at low concentration (Fig. 6). The [(Eu(TTA)3phen] luminescence intensity (613nm) decreased with increasing analyte solution concentrations in Fig. 6a, c, and d, from which the different quenching efficiencies of coumarin, tinidazole and AO can be found. In Fig. 6a and c, coumarin and tinidazole were more efficient than AO. With increasing AO concentration, the quenching of [(Eu(TTA)3phen] luminescence intensity was very slow indicated poor efficiency of AO for quenching. Additionally, higher AO concentrations led to increasing background fluorescence emission at 525 nm. To quantify the quenching trends for three drugs, we fit the emission intensity at 613 nm Fig. 6 b, d and f.

I0/I＝a·exp(K[C])＋b (1) where C is quencher concentration in mM, I and I0 are the luminescence intensities with and without quencher, a, b, and K are fitting constants. We measured variations in the emission intensity at 613 nm, and we found that the coumarin data could be fit: I0/I ＝9.4943 ·exp(1.5297[C]) － 8.8028 with a correlation coefficient of 0.9978 (Fig. 6b). Nonlinear Stern–Volmer plots might arise from ligand energy transfer or molecular collisions.36-37 Per Keizer, positive Stern-Volmer curvature can result at high analyte concentrations from static quenching or from rapid diffusion-limited quenching.38 To analyze tinidazole and AO luminescence quenching efficiency, we used a linear Stern–Volmer (S–V) equation I0/I＝1＋Ksv[C] (2) where C is quencher concentration, I and I0 are the luminescence intensities with and without quencher, and Ksv is a quenching constant (M-1).39

Figure 6 Solution-phase quenching of [(Eu(TTA)3phen] with coumarin, tinidazole, and AO. Variation of emission spectra of 0.1 mM [(Eu(TTA)3phen] solution (λex =365nm, pH 7.4) in the presence of increasing concentrations of (a) Coumarin, (c) Tinidazole, and (e) AO. Stern–Volmer plots for the quenching of 0.1 mM [(Eu(TTA)3phen] emission at 613 nm by (b) Coumarin, (d) Tinidazole (d) and (f) AO.


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We measured variations in the emission intensity at 613 nm, and we found that the I0/I ratio was linearly dependent on tinidazole and AO solution concentration (Fig. 6d and f). For tinidazole, the linear correlation could be fit to I0/I = 1.06 + 292.49[C], with a correlation coefficient of 0.9985. For AO, the linear correlation could be fit to I0/I = 1.02 + 4.23[C], with a correlation coefficient of 0.9981. We next verified that the same quenching effect could be observed for LC-doped crystals. Luminescent crystals incubated in 10mM coumarin, tinidazole, or AO solutions could be dramatically quenched in tens of minutes (Fig. 7). Slow quenching in AO and coumarin is consistent with the relatively large size of AO and coumarin compared to the solvent channels (Fig. 1). On the other hand, when the luminescent lifetime was measured (SI Fig. 8), acridine orange (AO) noticeably altered fluorescence lifetime. Hypothetically, small molecular drugs could be identified by measuring changes in luminescent lifetime as well as the crystal fluorescence quenching rate. The precise, regular structure of host crystals could provide unique opportunities to distinguish between small molecules via differences in intra-crystal transport.

Figure 7 Time-resolved microspectrophometer images for luminescent, LC-doped protein crystals in analyte solution excited at 365 nm (50 μm scale bar). (a)-(c) A luminescent, LC-doped protein crystal in coumarin solution. (d)-(f) A luminescent, LC-doped protein crystal in tinidazole solution. (g)(i) A luminescent, LC-doped protein crystal in AO solution.

Looking for existing related contributions40-43 we found a luminescent lanthanide metal–organic framework40-43, detection based on luminescent lanthanide complexes and signal amplification40-43 quantification of DNA based on Eu III luminescence40-43, and luminescent lanthanidefunctionalized gold nanoparticles.40-43 These prior examples establish that luminescent LC are useful materials in drug sensing applications (via quenching). The current work differs in that our LC are present as inclusions within biomolecular crystals, and in that the crystals simultaneously impact strong luminescence and excellent biocompatibility. The host crystal provides significant advantages, avoiding poor solubility, avoiding poor dispersion in many solvents, and preventing uncontrolled LC aggregation. In the long term, it may also be easier to retain LC inside a solid macroscopic particle that can be exposed to biological fluids, whereas LC itself in solution that would be lost upon attempts to reuse the sensor. Herein, the LC-doped protein crystals can be repeatedly subjected to new solution conditions.

To investigate the luminescent quenching mechanism of [(Eu(TTA)3phen] by coumarin, tinidazole, and AO, the lifetime of those mixed solutions was studied. Dynamic quenching is due to collisions between the fluorescent LC and the quencher molecules, whereupon the LC loses energy through a non-radiative pathway. In contrast to collision quenching, static quenching is due to theformation of non-fluorescent complexes between fluorescent analyte and quencher. We sought to distinguish the two quenching routes by measuring luminescence lifetime. In principle, static quenching will decrease the fraction of active LC without decreasing the luminescence lifetime for LC that are active. In contrast, collision-based quenching can reduce the excited state lifetime. The luminescence lifetimes of [(Eu(TTA)3phen] with 0.1 mM coumarin (448 μs) and 0.005 mM tinidazole (459 μs) (Supplementary Figure S8) was similar to the original solution (453 μs), consistent with static



quenching for tinidazole and coumarin at these concentrations. The luminescence lifetime of [(Eu(TTA)3phen] with AO, even at a low AO concentration of 0.01 mM, was much shorter than original solution (207 μs) which is consistent with dynamic quenching by AO.

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Conclusions

We grew small, high-aspect ratio HEWL crystals using an optimized batch recipe. The macroscopic quality of the HEWL crystals, as assessed using scanning electron microscopy, was still high after washing, crosslinking, and guest molecule soaking steps. The emission spectrum and spatial distribution of the [(Eu(TTA)3phen] within the HEWL crystals were revealed by microspectrophometer and laser scanning confocal microscopy, respectively. These tests established that protein crystals could be uniformly doped by [(Eu(TTA)3phen]. HRTEM and EDX measurements further identified [(Eu(TTA)3phen] nanocrystal lattice fringes and Eu peaks respectively when doped crystals were ground to powder. For future applications of these materials, perhaps the most interesting outcome was the observation that luminescent protein crystals could be quenched by coumarin, tinidazole and acridine orange. Quenching trends could be precisely fit using conventional Stern–Volmer analysis. In summary, this work provides a protocol for stepwise, in-crystal synthesis of luminescent guest molecules. Depending on the nature of the functional guest molecules, crystals in this class doped with fluorescent, analyte-sensitive, or catalytic guest molecules may find application as in optical sensing and catalysis.

Conflicts of interest

There are no conflicts of interest to declare.

Acknowledgements

This work was supported by (1) State Key Project of International Cooperation Research (2016YFE0110800, 2017YFE0108300); (2) National Natural Science Foundation of China (51373081, 51473082, 51703104; (3) the National Program for Introducing Talents of Discipline to Universities (“111” plan); (4) 1st class discipline program of Materials Science of Shandong Province.


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For Table of Contents Use Only

Drug Sensing Protein Crystals Doped with Luminescent Lanthanide Complexes Guotao Suna, Jianguo Tang*a, Christopher D. Snow*b, Zhenhua Lia, Yu Zhanga, Yao Wanga, Laurence A. Belfiore b a Institute of Hybrid Materials, National Center of International Research for Hybrid Materials Technology, National Base of International Science & Technology Cooperation, College of Materials Science and Engineering, Qingdao University, Qingdao, 266071, P. R. China. Email: [email protected] b Department of Chemical and Biological Engineering, Colorado State University, Fort Collins, Colorado 80523 USA. Email: [email protected]

Graphic:

This work provides a protocol for stepwise, in-crystal synthesis of luminescent guest

molecules. Luminescent protein crystals could be quenched by coumarin, tinidazole and acridine orange.

Synopsis ： We grew small, high-aspect ratio HEWL crystals using an optimized batch recipe. The emission spectrum and spatial distribution of the [(Eu(TTA)3phen] within the HEWL crystals were revealed. These tests established a protocol for stepwise, in-crystal synthesis of [(Eu(TTA)3phen]. Luminescent protein crystals could be subsequently quenched by coumarin, tinidazole and acridine orange.

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Drug Sensing Protein Crystals Doped with Luminescent Lanthanide

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