The Marriage of Protein and Lanthanide: Unveiling a Time-Resolved

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The Marriage of Protein and Lanthanide: Unveiling A Novel Time-Resolved Fluorescence Sensor Array Regulated by pH Toward High-Throughput Assay of Metal Ions in Biofluids Zi-Yang Lin, Zhi-bei Qu, Zi-Han Chen, Xin-Yue Han, Ling-Xue Deng, Qingying Luo, Zongwen Jin, Guoyue Shi, and Min Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01879 • Publication Date (Web): 01 Aug 2019 Downloaded from pubs.acs.org on August 2, 2019

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Analytical Chemistry

The Marriage of Protein and Lanthanide: Unveiling A Novel TimeResolved Fluorescence Sensor Array Regulated by pH Toward HighThroughput Assay of Metal Ions in Biofluids Zi-Yang Lin1, Zhi-bei Qu2, Zi-Han Chen1, Xin-Yue Han1, Ling-Xue Deng1, Qingying Luo3, Zongwen Jin3, Guoyue Shi1, Min Zhang* 1School

of Chemistry and Molecular Engineering, Shanghai Key Laboratory for Urban Ecological Processes and EcoRestoration, East China Normal University, 500 Dongchuan Road, Shanghai 200241, China. Email: [email protected] 2Joint Research Center for Precision Medicine, School of Chemistry and Chemical Engineering and School of Medicine, Sixth People’s Hospital South Campus, Shanghai Jiao tong University, Shanghai 200240, China. 3Research Center for Micro/Nano System & Bionic Medicine, Institute of Biomedical & Health Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, 1068 Xueyuan Avenue, Shenzhen, 518055, China. ABSTRACT: A protein/lanthanide complex (BSA/Tb3+)-based sensor array in two different pH buffers has been designed for highthroughput recognition and time-resolved fluorescence (TRF) detection of metal ions in biofluids. BSA acted as an antenna ligand can sensitize the fluorescence of Tb3+ (i.e. antenna effect), while the presence of metal ions would lead to the corresponding conformational change of BSA for altering the antenna effect accompanied with a substantial TRF performance of Tb3+. This principle has also been fully proved by both experimental characterizations and coarse-grained molecular dynamics (CG-MD) studies. By using Tris-HCl buffer with different pHs (at 7.4 and 8.5), 17 metal ions have been well distinguished by using our proposed BSA/Tb3+ sensor array. Moreover, the sensor array has the potential to discriminate different concentrations of the same metal ions and a mixture of metal ions. Remarkably, the detection of metal ions in biofluids can be realized by utilizing the presented sensor array, verifying its practical applications. The platform avoids the synthesis of multiplex sensing receptors, providing a new method for the construction of convenient and feasible lanthanide complex-based TRF sensing arrays.

Introduction

output.13 Despite the tremendous amount of work regarding lanthanide-complex probes, only a few complexes using protein as the antenna ligands which could absorb the excitation light and transfer it to the Tb3+. Jin et al. have utilized the Tb3+ as fluorescent probe to study the calcium-binding sites on bovine serum albumin (BSA).15 They reported that the TRF of Tb3+ is enhanced markedly when bound to BSA and the nonradiative energy transfer occurs from tryptophan (Trp) residues to the bound Tb3+. It is also reported that BSA can combine with some substances such as metal ions through its carboxyl side groups of glutamic acid (Glu) and aspartic acid (Asp), which could lead to the corresponding conformational change of BSA.15 Since BSA have different affinities toward various metal ions, metal ions-involved effects on the interaction between Tb3+ and BSA would regulate the diverse TRF performances of Tb3+. In consideration of these findings, the TRF probes based on the marriage of BSA and Tb3+ could be thus designed to detect different kinds of metal ions. Currently, a series of methods have been reported for the detection of metal ions, including instrument-based standard assays, e.g. atomic absorption spectrometry (AAS). Compared with that, in recent years, array-based sensors using a group of sensing receptors has emerged as an alternative powerful tool for the determination of multiple metal ions.16,17,18,19,20,21,22 For example, Chang and co-workers realized the detection of 7 metal ions by applying a sensor array made up of 5 small

Time-resolved fluorescence (TRF) techniques with the extremely long fluorescence lifetimes of lanthanide complexes have attracted great attention from scientists.1,2 TRF measurements can eliminate the interferences from background fluorescence of biological media by setting a delay time between the excitation pulse and the fluorescence determination, therefore allowing the fluorescence of the biological media to decay before measuring that of the probe.3 Lanthanide (mainly Tb3+) complexes, possessing super long-lived fluorescence with large Stokes shifts and sharp emission profiles, are ideally suitable for such TRF measurements applications.4,5 Typically, lanthanide ions have low fluorescence since the direct excitation of them is relatively difficult due to Laporteforbidden f-f transitions.6 Thus, chromophore-containing (“antenna”) ligands must be coordinated to lanthanide ions (such as Tb3+) to enhance their fluorescence owing to the energy transfer from the ligand’s triplet excited state to the emitting electronic level of lanthanide ions, which is known as the antenna effect.7,8 Examples of these ligands are lanthanide chelates, cryptates, lanthanide-binding peptides, sequencespecific DNA, etc. Due to the characteristic sensitizing process, Tb3+ and its complexes have been widely used as fluorescence sensing probes.9,10,11,12,13,14 For example, Nie et al. developed a TRF biosensor for sensing enzymatic activity by using guanine (G)-rich DNA-sensitized Tb3+ fluorescence as the signal 1

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fluorophores units and a metal chelator.22 However, these arraybased sensors for massive applications are easily limited by the complex synthesis of multiple sensing units. To solve this problem, multidimensional sensing platforms were gradually developed which could extract multidimensional signals from a single sensing receptor.23,24 Therefore, much attention has been paid to constructing a multitarget detection system with a single signal-source. Inspired by the above facts, herein, we present a novel TRF sensing platform via the combination of BSA with Tb3+, in which BSA can act as an antenna ligand to sensitize the TRF of Tb3+ (i.e., antenna effect). Interestingly, BSA/Tb3+ shows different TRF performances in Tris-HCl buffer with various pHs, which makes it possible to design a pH-regulated BSA/Tb3+ sensor array to realize the detection of multiple targets. The addition of metal ions to BSA/Tb3+ can lead to the corresponding conformational change of BSA, further cause the alteration of the antenna effect accompanied with the substantial TRF performance of Tb3+. Therefore, utilizing the pH-dependent properties of BSA/Tb3+, we construct a BSA/Tb3+-based sensor array for discrimination of various metal ions by using Tris-HCl buffer with different pHs (at 7.4 and 8.5) to regulate the TRF performances of BSA/Tb3+. Our strategy avoids the complicated synthesis of multiplex receptors and exhibits the advantages to extract multiple information of TRF property from a single material of BSA/Tb3+. The selected BSA/Tb3+ sensors can exhibit varying TRF responses due to their diverse affinities towards different metal ions. Principal component analysis (PCA) was used to analyze these TRF response patterns.25 Our experiments show that the proposed pH-regulated BSA/Tb3+ sensor array can be applied for discrimination of 17 different metal ions in aqueous buffer. Furthermore, the BSA/Tb3+ sensor array has been employed to identify metal ions in artificial biofluids to verify its practical applications, and the results are well in line with that of standard ICP-AES method, confirming the high accuracy and reliability of the proposed sensor array.

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are of analytical reagent grade. All solutions were prepared using ultrapure water from a Milli-Q ultrapure water system. Instrumentation. Fluorescence measurements were performed on the TECAN microplate reader using a black 384 well microplate. For fluorescence measurement, the excitation wavelength used was 284 nm. For the TRF spectra, a delay time of 50 μs and a gate time of 2000 μs were used. The fluorescence lifetimes spectra were recorded with an Edinburgh fluorescence spectrometer (FLS980), in which the pulse width of the excitation source was 810.7 ps and the bandwidth was 10.6 nm. Zeta potentials were measured using a Malvern Zetasizer Nanoseries. Circular dichroism (CD) measurements were carried out using a Chirascan CD spectrometer (Applied Photophysics). The CD spectra were collected at room temperature over the range 180-260 nm using a quartz SUPRASIL with an optical path of 10 mm. BSA-Sensitized the TRF of Tb3+. Different concentrations of BSA and Tb3+ (1 mM) were prepared in Tris-HCl buffer (10 mM). The mixture was incubated for 6 min at room temperature. Next, the TRF spectra of BSA/Tb3+ was recorded when excited at 284 nm. The optimal concentration of BSA was investigated. Discrimination of Metal ions by Using BSA/Tb3+ Sensors. 0.2 mg/mL BSA was first mixed with 1 mM Tb3+ in 10 mM Tris-HCl buffer with two different pHs (7.4 and 8.5) for 6 min as a BSA/Tb3+ sensor array. Next, 10 μL each metal ions (Cu2+, Co2+, Zn2+, Mn2+, Ni2+, Pb2+, Ag+, Li+, Na+, Fe3+, Ca2+, Mg2+, Al3+, K+, Cd2+, Cr3+ and Hg2+) was mixed with 90 μL BSA/Tb3+ sensors and then incubated for 12 min at room temperature. Finally, the TRF intensities of the obtained mixtures were measured using the TECAN microplate reader. The TRF responses, i.e. [(F0-F)/F0], were calculated and used for pattern recognition and discrimination analysis, where F and F0 are the TRF responses of BSA/Tb3+ at 548 nm in the presence and absence of metal ions. We generated the TRF responses five times for each metal ion against the pH-regulated BSA/Tb3+ sensors. The TRF-responses patterns (2 BSA/Tb3+ sensors × 17 metal ions × 5 replicates) was obtained and analyzed with PCA to identify the tested metal ions. Computational simulations and investigations. Molecular dynamics (MD) simulations were performed with the GROMACS package version 2019 beta1,26 with the Martini v2.2 force field parameters,27 and standard simulation settings. The BSA structure was imported from RCSB protein data bank (PDB) website (pdb ID: 4f5s) (Figure S1).28 The structure was further coarse-grained (CG) to reduce the computational cost, in solvation box using polarized CG water molecules. The hydrated Tb3+ was adapted from previous report.29 The molecular ratio of Tb3+ and BSA protein was set to be 1000:1. The extra charges were balanced with chloride ions (Cl-). After initial energy minimization with position restraints applied on the protein beads, short equilibrium processes were performed with the position restraints applied to the protein backbone beads. All simulations were performed with a 10-fs time step, a temperature of 300 K set, with velocity-rescaling thermostats,30 with a time constant for coupling of 1 ps. An isotropic pressure of 1 bar maintained with the Berendsen barostat,31 with a compressibility of 3×10-4 bar-1, and a relaxation time constant of 5 ps. Production runs of the duration last for 10 µs to get the final data.

Scheme 1. BSA/Tb3+-Based Sensor Array for Pattern Discrimination of Metal Ions.

Experimental Section Materials. Bovine serum albumin (BSA) was purchased from Sangon Inc. (Shanghai, China). Tb(NO3)3 was purchased from Diyang Chemical (Shanghai) Co. Ltd. All other reagents 2


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Analytical Chemistry

Results and Discussion

Figure 1. (a) TRF spectra of Tb3+, BSA/Tb3+; (b) Fluorescence lifetimes of Tb3+, BSA/Tb3+, BSA/Tb3++Cu2+, and BSA/Tb3++Cd2+; (c) TRF responses of Tb3+, BSA/Tb3+ to TrisHCl buffer with various pHs; (d) Zeta potentials of BSA/Tb3+ in Tris-HCl buffer with various pHs.

Intrinsic fluorescence of BSA was measured by excitation of its Trp residues at ~280 nm, and the presence of a strong emission peak at ~330 nm (Figure S2). BSA has a UV absorption band around 250-300 nm (Figure S3), which can make it as an 'antenna' to absorb incident light (~280 nm) and promote the nonradiative energy transfer from Trp residues to the bound Tb3+ (i.e. antenna effect, Figure S4). The TRF spectrum of Tb3+ and BSA/Tb3+ were measured in aqueous solutions at room temperature and the results are presented in Figure 1a. Tb3+ alone showed very weak TRF when excited at a wavelength of 284 nm. While the aqueous solution of BSA/Tb3+ produced a significant enhanced TRF emission peak around 492 nm, 548 nm, 589 nm, and 622 nm upon illuminated at 284 nm, indicating that BSA can sensitize Tb3+ to fluorescence. The fluorescence lifetime was then measured (Figure 1b), and the corresponding curve fit traces were also presented (Figure S5). The long fluorescence lifetime of BSA/Tb3+ make it an ideal tool to background-free detect target analytes using TRF technique (Figure S6). With increasing pH varying from 4 to 10, the fluorescence emission intensity of BSA/Tb3+ at 548 nm continuously increases (Figure 1c), suggesting that the BSA/Tb3+ is potential to be used as a pH indicator. As a control, the TRF of Tb3+ at 548 nm in the absence of BSA almost remains constant with increasing pH indicated. Thus, the pH-dependent property of BSA/Tb3+ can be ascribed to the conformation alteration of BSA in various pHs. To be more specific, the major groups in BSA bound to Tb3+ are the carboxyl side chains of glutamic acid (Glu) and aspartic acid (Asp) residues,15 which make the interaction between Tb3+ and BSA changed by utilizing pH to control the state of the carboxyl side. Thus, the TRF properties of BSA/Tb3+ can be further changed in different pHs. As shown in Figure S7, circular dichroism (CD) measurements were carried out to represent the conformational change of BSA/Tb3+ in Tris-HCl buffer with two pHs (at 7.4 and 8.5). The zeta potentials were also measured in Tris-HCl buffer with different pHs to help understand the pHdependent TRF properties of BSA/Tb3+. From Figure 1d, the potentials of BSA/Tb3+ decreased with the increase of pH since the acidic and basic residues in BSA are protonated/deprotonated with pHs. The results indicate that pH could influence the structure of BSA/Tb3+, which was consistent with the above discussion.

As indicated above, BSA/Tb3+ exhibits enhanced TRF owing to the energy transfer from BSA to Tb3+ (i.e. antenna effect). Also, it was reported that BSA can combine with metal ions through their carboxyl group of Asp and Glu, which could induce the conformational change of BSA/Tb3+ complexes owing to the high binding affinity between the BSA and metal ions. For each metal ion, BSA/Tb3+ shows various kinds of TRF responses due to the different affinities between metal ions and BSA. In addition, BSA/Tb3+ can be regulated by pH to display different TRF performances, indicating that the highthroughput discrimination of metal ions shall be possible through a sensor array of pH-involved BSA/Tb3+. For each metal ion sample, the BSA/Tb3+ sensor array generates a distinct TRF response pattern that can be further differentiated by PCA. In this work, as a proof of concept, BSA used as an antenna ligand for Tb3+ to build a pH-regulated BSA/Tb3+ sensor array (pH at 7.4 and 8.5), realizing the pattern discrimination of metal ions, which provides a novel sensor platform for the detection of metal ions. Fluorescence titrations experiment were first conducted to investigate an optimum BSA concentration for effective sensitizing the TRF of Tb3+. Figure S8 shows the TRF responses of BSA/Tb3+ when introducing different concentrations of BSA into Tb3+ solution in Tris-HCl buffer. With the increase of the BSA concentration, the most intense emission peak of Tb3+ at 548 nm gradually increased to the peak at 0.2 mg mL-1, where the quality for sensitizing the TRF of Tb3+ reached the maximum. Therefore, BSA (0.2 mg mL-1) was picked in the following experiments. The TRF at 548 nm of Tb3+ increased with increasing reaction time and reached a saturation point at 6 min (Figure S9). Thus, a reaction time of 6 min for Tb3+ and BSA was chosen in the following experiments. Though the sensitization of the TRF of Tb3+ by BSA was discovered decades ago, the combination details of Tb3+ and BSA remain unclear, such as specific recognition sites, molecular ratio, energy transfer process, etc. Tb3+ has multiple affinity to protein because of the ability to form coordination bonds to various functional groups, including carboxyl, hydroxyl, and amines, as well as the strong electrostatic charges. Jin et. al. assumed that Tb3+ combined onto BSA through Asp and Glu residues via fluorescence titration.15 However, their results were lack of quantitation significance, and we still do not know how these amino acid residues combine with Tb3+, and neither the stoichiometric proportion. Herein, we applied molecular dynamics simulations to better understand the combination of Tb3+ and BSA in our study (Figure 2). A coarsegrained molecular dynamic (CG-MD) method was employed to accelerate the computation. We found that BSA did not exhibit obvious configuration change immerging in Tb3+ solution. It is observed that there are approximately 69 Tb3+ attached to 1 BSA, determined by a distance threshold of 0.7 nm between the ion and the protein beads. A sum of 108 amino acid residues were involved in the combining process, including 30 Glu, 27 Asp, 11 Ala, and 8 Trp. The rest residues are random and 3



dynamic combinations that frequently occur. The major combining sites are Glu and Asp, which is consistent with our perspective.

Figure 2. Illustration of BSA and the combined Tb3+ from the CG-MD simulations. Yellow and white balls represent BSA residue and backbone particles. Green balls represent combined Tb3+. Waters, non-combined ions and countered Cl- were invisible to make the image clear. In this work, the multiple readouts for constructing a sensor array originated from the TRF signals of BSA/Tb3+ at different pH buffers. We chose the TRF changes at two pH buffers (7.4 and 8.5) as receptors for straightforward data acquisition. Preliminary studies were performed in aqueous solution. 17 metal ions, including Cu2+, Co2+, Zn2+, Mn2+, Ni2+, Pb2+, Ag+, Li+, Na+, Fe3+, Ca2+, Mg2+, Al3+, K+, Cd2+, Cr3+ and Hg2+ (each at 50 μM), were mixed with BSA/Tb3+ sensor array and incubated for 12 min (Figure S10). A 50 μs delay time and a 2000 μs gate time were used for the TRF spectra, the TRF changes at 548 nm were recorded. The diverse responses of the sensor array to different metal ions were shown in Figure 3. Five replicates were measured for each metal ion, generating pattern [(F0-F)/F0] data. The response data including 170 data points (2 receptors × 17 metal ions × 5 replicates) was obtained for generating corresponding heat map, which was further analyzed with PCA. From Figure 3a, we can see clearly that BSA/Tb3+ sensors exhibit different response mode to 17 metal ions according to the TRF changes [(F0-F)/F0]. It was shown that TRF quenching of BSA/Tb3+ occurred in the presence of metal ions, except in the case of Zn2+ and Cd2+. The TRF changes [(F0-F)/F0] was negligible in the case of Pb2+, Li+, Na+, Ca2+, Mg2+, Al3+, K+, and Hg2+, whereas it was slightly more significant in the presence of Ag+. In contrast, strong TRF responses were observed in the presence of Cu2+, Co2+, Mn2+, Ni2+, Fe3+ and Cr3+. As mentioned above, BSA can interact with metal ions through their carboxyl group of Asp and Glu, which may cause the conformational altering in BSA molecular. Therefore, it is rational to consider that metal ions binding to BSA may affect the interaction of Tb3+ with BSA in the above system and further influence the TRF responses of BSA/Tb3+.

Figure 3. (a) The patterns of the 17 metal ions based on the TRF changes [(F0-F)/F0]; (b) Heat map based on the TRF patterns of BSA/Tb3+ sensor array; (c) PCA plot for the discrimination of 17 metal ions (50 μM). For each metal ion, the different affinity toward BSA make the sensor array generate a unique TRF response pattern that is further differentiated via PCA. PCA concentrates the most significant characteristics (variance) of the data into a lower dimensional space. In our test, the data matric was transformed into three principal components. After the analysis, the first two canonical factors (89.112 and 10.888%) were employed to produce a two-dimensional (2D) plot. As shown in Figure 3c, 17 separate clusters corresponding to 17 metal ions demonstrated the metal ions were identified from each other. Meanwhile, the short distances between the five replicated points in each cluster illustrated high accuracy and satisfactory reproducibility of the BSA/Tb3+ sensor array. The above results suggested that the BSA/Tb3+ sensors have strong discrimination ability of detecting 17 metal ions. To make the method more applicable in real situation, lower concentrations of 17 metal ions (each at 10 μM, 1 μM) were also tested using the BSA/Tb3+ sensor array (Figure S11 and S12). From Figure S7a and S8a, 17 metal ions at such lower concentrations can also formed tight clusters with a well separation between each other. This means that the proposed BSA/Tb3+ sensor array has great potential in detecting metal ions in practical usage. To further explore the possible mechanism involved in this system, spectroscopic measurements were carried out to characterize the circular dichroism (CD), absorption and fluorescence of BSA in the absence or presence of Tb3+ and/or other metal ions (Figure S13, S14, and S15). The CD spectrum in the far-UV region (180-250 nm) is widely used to characterize the secondary structure, conformation, and the stability of proteins in solution.32 Therefore, it is an important tool for speculating the conformational changes of BSA after adding different metal ions. The CD spectra of BSA in Tris-HCl buffer at two pHs (7.4 and 8.5) in the absence and presence of Tb3+ and/or various metal ions were recorded. We took pH at 4


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Analytical Chemistry 7.4 as an example. As shown in Figure S13, one positive band and two negative bands observed in the CD spectra of BSA in Tris-HCl buffer (pH 7.4) at 192 nm, 208 nm, and 222 nm in the UV region respectively were characterized as the typical α-helix structure of protein. The negative bands at 208 nm and 222 nm correspond to the n→π* transition due to the peptide bond of an α-helix.33 Upon the addition of Tb3+ into the BSA solution, two new positive sharp peaks at 196 and 198 nm accompanied by two negative peaks at 200 and 206 nm appeared. The two positive peaks at 196 and 198 nm originate from π → π* transition in the peptide bond and reflect the characteristic signal of the β-sheet conformation of the protein. Further, the influence of various metal ions (Cu2+, Co2+, Ag+, Al3+, Zn2+, and Cd2+) on the conformational change of BSA/Tb3+ was investigated using the CD spectra. We observed a significant increase in the intensity of α-helix peaks at around 193 nm upon the addition of Cu2+ or Co2+ (Figure S13a, S13b), whereas the intensity of two positive peaks at around 197 and 199 nm ascribed to the β-sheet structure showed a gradual declination. In contrast to Cu2+, a new positive peak at around 195 nm was observed in CD spectra when adding Zn2+ or Cd2+ (Figure S13e, S13f). Compared with BSA/Tb3+, the negative peak at 206 nm was not detected in the presence of Ag+ (Figure S13c), which reveals a substantial change in α-helix content. A new positive peak at 206 nm was observed in the presence of Al3+ (Figure S13d), suggesting the β-turn structure in BSA. Besides, the presence of metal ions (e.g. Na+, Cu2+, Cd3+ or Ag+) to BSA/Tb3+ can alter its absorption (Figure S14) and fluorescence (Figure S15a), leading to the metal ion-regulated antenna effect of BSA toward the TRF of Tb3+ (Figure S15c). For example, upon the addition of Cu2+, the absorption of BSA/Tb3+ was changed and its fluorescence quenched at 330 nm (Figure S15b), followed by the decrease of the TRF of Tb3+ at 548 nm (Figure S15d). The above results indicate that conformational changes of BSA were observed after adding various metal ions due to their molecular interactions for altering the antenna effect (Figure S4), accompanied with significant pattern differences in TRF intensities. We experimentally found that various metal ions have distinguishable TRF-response patterns of BSA/Tb3+. For the transition metal ions that have UV-vis absorption activity, such as Ag+, Fe3+, Cu2+, and so on, their quenching effect can be simply attributed to the photo-induced electron transfer (PET) from Tb3+ to the valence orbitals of the transition metals. However, for the non-transition metals such as Na+, K+, Ca2+, they also showed specific TRF responses to BSA/Tb3+. This phenomenon cannot be explained by regular PET or fluorescence resonance energy transfer (FRET) mechanisms. Another series of CG-MD simulations were further performed to study the influence of the non-transition metals to BSA/Tb3+. Na+ was chosen to be the representative model of non-transition metals, since it is the only one that has accurate hydrated force field parameters in CG method. The molecular ratio of Na+ over Tb3+ was built as 1:20, corresponding to the realistic experiment conditions. It was observed that, in the presence of Na+, the combined Tb3+ increases by 15% (Figure S16). The rise of combined Tb3+ possibly results from the change of the BSA conformation caused by Na+. It is known that the final fluorescent intensity is largely determined by the FRET efficiency E (Equation 1) from the Trp residues to combined

Tb3+. The characteristic distance d0 from Tb3+ and Trp residues were calculated (Equation 2). 𝟏

𝑬= 𝟏+

(

𝒅

)

𝟏

𝒏

𝒅𝟎 =

(

(1)

𝟔

𝒅𝟎

∑𝒊 (𝒅𝒊) ―𝟔 𝒏

)

―𝟔 (2)

In the presence of Na+, the characteristic distance from Tb3+ to Trp residues increases from 2.40 nm to 2.47 nm, which leads to a drop of TRF intensity to 84.5% of the original one. The final TRF intensity was determined by the addition of the number change of combined Tb3+ and the efficiency change of the FRET process. The theoretical TRF of BSA/Tb3+ challenged with Na+ from the CG-MD simulations were calculated as 97.2% of the original intensity, which shows similar correspondence to the experimental measure (98.8%). Thus, the influence of various metal ions to the TRF of BSA/Tb3+ can be attributed to three factors: (a) PET process from Tb3+ to transition metals; (b) numbers of Tb3+ combined onto BSA, which may result from the hydration effects of metal ions on BSA conformations; (c) the change of average distance between Tb3+ and Trp residues, leading to a change in FRET efficiency. After successful discrimination of the 17 metal ions, the next step was to investigated the response of sensor array to specific metal ions with different concentrations. Cu2+, Co2+ and Fe3+, which play essential roles in biochemistry, were chosen as the model analytes. Those heavy metal ions are also the source of many major diseases, such as Alzheimer’s diseases.34 In this regard, accurate and rapid detection/identification of these metal ions are of great significance for human health. In this work, different concentrations of Cu2+, Co2+ and Fe3+ were separately treated with the BSA/Tb3+ sensor array (Figure 4, S17-S19). As shown in Figure 4b, e, f, the TRF changes [(F0F)/F0] of the sensor array grow with the increase of metal ions’ concentration. From Figure 4a, d, g, we found that the PCA plots were not random, but rather followed certain patterns, which can be differentiated from each other at many concentrations. Meanwhile, PC1, known as the first discriminant factor, can be simply used to quantify the concentrations of metal ions, since the PC2 (the second discriminant factor) was not exceeding 40% (Figure 4c, f, i). PC1 value (y) versus the logarithm of Cu2+ concentration (x) was fitted as y=1.665x-0.4956 with the relative coefficient (R2) of 0.9818, showing a linear detection range from 0.1 to 50 μM with a limited detection of 8.06 nM, which is significantly lower than the normal levels of Cu2+ in drinking water (20 μM) and individual blood (11.8-23.6 μM).35 Similarly, the calibration curve for Co2+ was y=1.715x-0.6626 with R2 of 0.9897, the calibration curve for Fe3+ was y=2.139x1.228 with R2 of 0.9515. The limits of detection for Co2+ and Fe3+ were 7.82 nM and 6.27 nM, respectively.

5



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example, biofluids (including saliva, urine and sweat), play an essential role in health. Namely, it is a sample taken for symptomatic examination or evaluation, and for identification of diseases in medical contexts. Hence, we prepared a series of artificial biofluids, such as urine, saliva and sweat to illustrate the practical use of the BSA/Tb sensor array. Then the two biofluid-derived metal ions (i.e., Cu2+ and Fe3+), whose uncommon contents in biofluids can be closely linked to various neurodegenerative diseases, were chosen as analytes, spiking into urine and sweat respectively with different kinds of concentrations (Table 1 and Table S1). For example, the progressive hepatolenticular degeneration, or Wilson’s disease, is a genetic disorder of copper metabolism.35 In Wilson’s disease, the 24-h urinary copper excretion is increased, and the concentration taken as suggestive of disease is greater than 1.6 μmol per 24h.36 And 0.6 μmol per 24h is taken as the reference limit for normal 24-h excretion of copper. As we all know, the normal adults have a urine output of 1000-2000 mL per 24h. Consequently, the normal level of copper is around 0.6 μM. As for people who suffer with Wilson’s diseases, more than 1.6 μM will be mostly be found in urine. Based on these facts, we spiked 0.5 μM as normal and 2, 5 μM as abnormal into urine samples to illustrate the BSA/Tb3+ sensor array for evaluating the abnormality of urine. As shown in Table 1, the recoveries of Cu2+ are varied from 103.0 to 117.0% in artificial urine. The results of these spiked concentrations of Cu2+ were in good agreement with results obtained by the traditional ICP-AES methods, indicating the potential applications for detection of metal ions in the complex environments and diagnosis of abnormal enhancement of metal in biofluids.

Figure 4. Discrimination of Cu2+, Co2+, and Fe3+ at different concentrations. (a, d, g) PCA plots for the detection of metal ions in different concentrations; (b, e, h) The pattern of the metal ions based on the TRF changes (F0-F)/F0; (c, f, i) The relationship between PC1 and different concentrations of metal ions. As we had obtained differentiation at various concentrations of metal ions, we investigated the discrimination ability of BSA/Tb3+ sensor array toward the mixtures of metal ions. A mixture of different valence states of Fe (Fe3+ and Fe2+), Cr (Cr3+ and Cr6+) were chosen as analytes. For example, the mixture of Fe3+ and Fe2+ with different molar ratios (Fe3+/Fe2+=20/0, 16/4, 12/8, 8/12, 4/16, and 0/20, 20 μM in total) was subsequently tested using the sensor array (Figure 5, S20 and S21). As shown in Figure 5a, these mixtures, as well as pure Fe3+ and Fe2+, were clearly separated from each other in PCA plot and properly arranged with the order of molar ratios. The mixture of Cr (Cr3+ and Cr6+) was also distinguished from each other using the BSA/Tb3+ sensors (Figure 5b). The above results illustrated that this system can be extremely sensitive to the valence states of metal ions, which would be potentially fit for determination of complex composition.

Table 1. Detection of Cu2+ in Urine Using BSA/Tb3+ sensor array. Entry*

Urine

Actual (μM) 0.5 1 2 5

ICP-AES (μM) 0.47 0.99 1.99 5.27

BSA/Tb3+ (μM) 0.55 1.17 2.15 5.15

Recovery (%) 110.0 117.0 107.5 103.0

RSD(%) 2.31 2.19 1.47 1.36

To evaluate the discrimination ability of BSA/Tb3+ sensor array toward biofluid-relevant metal ion mixtures, several mixing groups were set in artificial urine and saliva respectively: (1) 10 μM Cu2+ + 0 μM Fe3+; (2) 6 μM Cu2+ + 4 μM Fe3+; (3) 4 μM Cu2+ + 6 μM Fe3+; (4) 0 μM Cu2+ + 10 μM Fe3+. In addition, three test samples were prepared in urine and saliva as a comparison: (A) 8 μM Cu2+ + 2 μM Fe3+; (B) 5 μM Cu2+ + 5 μM Fe3+; (C) 2 μM Cu2+ + 8 μM Fe3+. From Figure 6, PCA plot for Cu2+/Fe3+ with various molar ratios were followed certain patterns. And the test samples (A, B and C) can be clearly observed in the right position matching their molar ratios. For example, sample B was located among the two standard mixtures with Cu2+/Fe3+ molar ratios at 6:4, and 4:6, indicating the concentration of Cu2+ were from 4 μM to 6 μM and of Fe3+ were from 4 μM to 6 μM in artificial urine, which is in line with the results detected by traditional ICP-AES methods. Therefore, the above results illustrated that the proposed BSA/Tb3+ sensor array could effectively detect metal ions in biofluid-relevant mixtures with high accuracy. Figure S22 shows the detection of biofluid-relevant mixtures Cu2+/Fe3+ in the artificial saliva using BSA/Tb3+ sensors, proving the capacity of BSA/Tb3+ sensors for semi-quantitatively sensing metal ion mixtures in biofluids.

Figure 5. PCA plots for the BSA/Tb3+ sensor array against the mixtures of metal ions. (a) Fe3+ and Fe2+; (b) Cr3+ and Cr6+. To demonstrate the effectiveness of BSA/Tb3+ sensors, the proposed method is applied in the detection of metal ions in complex environments by detecting recoveries after adding given amounts of metal ions. Complex environments, for 6


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Analytical Chemistry biofluids demonstrates the practical application of our sensor array toward the non-invasive diagnosis of diseases.

Supporting Information Structure of original BSA; Excitation and emission spectra of BSA and Trp; Absorption spectrum of BSA; An illustration of the antenna effect; Fluorescence lifetime data with corresponding fitting curves; Scheme illustration of the TRF detection mode used in our work; Circular dichroism (CD) spectra of BSA/Tb3+ in two Tris-HCl buffer with varied pH; TRF responses of Tb3+ in the presence of different concentrations of BSA; The optimization of reaction time between Tb3+ and BSA, BSA/Tb3+ and Cu2+ in Tris-HCl buffer (pH=7.4); CD spectra of BSA, BSA/metal ions, BSA/Tb3+, BSA/Tb3+/metal ions in Tris-HCl buffer (pH=7.4); Absorption spectra of BSA/Tb3+ and BSA/Tb3+ respectively challenged with different metal ions; Fluorescence or TRF spectra of BSA, BSA/Tb3+, and BSA/Tb3+ respectively challenged with different metal ions; Illustration of the BSA protein and the combined Tb3+ in the presence of Na+ from the CG-MD simulations.; Heat map derived from the TRF responses pattern of BSA/Tb3+ sensors against Cu2+, Co2+, and Fe3+; TRF responses of BSA/Tb3+ sensors against the mixture of Fe2+/Fe3+ and Cr3+/Cr6+; PCA plot for the TRF pattern of BSA/Tb3+ sensors toward the mixture of Cu2+/Fe3+ in artificial saliva; Detection of Fe3+ in sweat using the BSA/Tb3+ sensor array; Comparison about sensitivity between this work and other fluorescent sensors.

Figure 6. PCA plot for fluorescence patterns of BSA/Tb3+ sensors toward the mixtures of Fe3+/Cu2+ in artificial urine. Table shows the performances of BSA/Tb3+ sensors compared with ICP-AES. The approach for the determination of metal ions (e.g. Cu2+ and Fe3+) is compared with previously reported fluorescent sensors from several respects, such as the linear detection range, the limit of detection (LOD), the species of metal ions, and their applications, as shown in Table S2 and Table S3. The proposed BSA/Tb3+ sensors demonstrate absolute superiority in terms of the LOD and the species of metal ions. More importantly, different from other fluorescent probes, BSA/Tb3+ sensor array avoids the complicated synthesis of multiplex receptors and exhibits the advantages to extract multiple information of fluorescence property from a single material of BSA/Tb3+.

AUTHOR INFORMATION Corresponding Author *Min Zhang, Email: [email protected] Notes The authors declare no competing financial interest.

Conclusion

ACKNOWLEDGMENT

In summary, we have developed an effective and highthroughput method for pattern recognition of metal ions using BSA/Tb3+ as receptor with multiple readouts for the first time. This novel strategy has several advantageous features compared with previous sensor arrays. The system is devised via the combination of BSA with Tb3+, in which BSA could sensitize the TRF of Tb3+ acted as an antenna ligand. Moreover, the pHdependent property of BSA/Tb3+ makes it possible to be the only sensing receptor, which obtain multiple readouts for array generation just regulating the TRF responses of BSA/Tb3+ by pH, without the need to synthesize various sensor units. By using Tris-HCl buffer with different pHs (at 7.4 and 8.5), the proposed BSA/Tb3+ sensor array can realize the TRF discrimination of 17 metal ions in aqueous buffer. Additionally, the sensor array has the potential to discriminate different concentrations of the same metal ions as well as a mixture of metal ions. This principle has also been fully proved by both experimental characterizations and coarse-grained molecular dynamics (CG-MD) studies. In addition, the resultant in

This work was supported by the National Natural Science Foundation of China (21775044, 21675053, 21635003), the Shanghai Science and Technology Committee (19ZR1473300, 19411971700, 18DZ1112700), Science and Technology Planning Project of Guangdong Province (2016B020238003) and the Fundamental Research Funds for the Central Universities.

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The Marriage of Protein and Lanthanide: Unveiling a Time-Resolved

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