Aggregation-Induced Emission Probe for Specific Turn-On

Subscriber access provided by READING UNIV

Article

AIE probe for specific turn-on quantification of sTfR: an important disease marker for iron deficiency anemia and kidney diseases Ruoyu Zhang, Simon H. P. Sung, Guangxue Feng, Chong-Jing Zhang, * Kenry, Ben Zhong Tang, and Bin Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03694 • Publication Date (Web): 06 Dec 2017 Downloaded from http://pubs.acs.org on December 6, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 16 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

Analytical Chemistry

AIE probe for specific turn-on quantification of sTfR: an important disease marker for iron deficiency anemia and kidney diseases Ruoyu Zhang,†,‡ Simon H. P. Sung,§,‡ Guangxue Feng,† Chong-Jing Zhang,† Kenry,† Ben Zhong Tang,§,∥,* and Bin Liu†,#,*

†

Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585

§

Department of Chemistry, Hong Kong Branch of Chinese National Engineering Research Centre for Tissue Restoration and Reconstruction, HKUST Jockey Club Institute for Advanced Study, Institute of Molecular Functional Materials, Division of Biomedical Engineering, State Key Laboratory of Molecular Neuroscience, Division of Life Science, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong ∥

SCUT-HKUST Joint Research Laboratory, Guangdong Innovative Research Team, State Key Laboratory of Luminescent Materials & Devices, South China University of Technology, Guangzhou 510640, China #

Institute of Materials Research and Engineering (IMRE), Agency for Science, Technology and

Research (A*STAR), 2 Fusionopolis Way, Innovis, Singapore 138634 ‡

These authors contributed equally to this work.

*

Corresponding author (E-mail: [email protected]; [email protected]; Fax: +65 6779 1936)

1 ACS Paragon Plus Environment

Analytical Chemistry 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

Page 2 of 16

Abstract Transferrin receptor (TfR) is overexpressed on the surface of many cancer cells due to its vital roles in iron circulation and cellular respiration. Soluble transferrin receptor (sTfR), a truncated extracellular form of TfR in serum, is an important marker of iron deficiency anemia (IDA) and bone marrow failure in cancer patients. More recently, sTfR level in urine has been related to a specific kidney disease of Henoch–Schönlein purpura nephritis (HSPN). Despite of the universal significance of sTfR, there is still lack of a simple and sensitive method for the quantification of sTfR. Furthermore, it is desirable to have a probe which can detect both TfR and sTfR for further comparison study. In this work, we developed a water-soluble AIE-peptide conjugate with aggregation-induced emission (AIE) characteristics. Taking advantage of the negligible emission from molecularly dissolved TPE, the probe TPE-2T7 was used for the light-up detection of sTfR. The probe itself is nonemissive in aqueous solution, but it turns on its fluorescence upon interaction with sTfR to yield a detection limit of 0.27 µg/mL, which is much lower than the sTfR level in IDA patients. Furthermore, a proof-of-concept experiment validates the potential of the probe for diagnosis of HSPN by urine test. Key words: AIE (aggregation-induced emission), sTfR (soluble transferrin receptor), IDA (iron deficiency anemia), urine test.


Page 3 of 16 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


Introduction Transferrin receptor (TfR) imports iron by internalizing transferrin-iron complex through endocytosis. It plays an important role in iron circulation and cellular respiration. The level of TfR is upregulated when there is a higher proliferation rate or a lower iron concentration. TfR could be overexpressed on cancer cell surface, which may be up to a hundred-fold higher than the average TfR level on normal cells.1 TfR is regarded as an important disease marker for early diagnosis of cancer and identification of tumor stage.2,3 Basically, TfR is a cell-membrane glycoprotein, but it also presents in the circulation. The extracellular portion of TfR can be cleaved and released into serum. It is named as serum TfR (or soluble transferrin receptor, sTfR).4 In the early stage of iron-deficient condition, there is an increase in concentration of sTfR, prior to the development of anemia. More importantly, the sTfR level will not be affected by chronic inflammation. It has thus been regarded as a good marker in discriminating iron deficiency anemia (IDA) and anemia of chronic disease (ACD). 5,6 In addition, assessment of sTfR level provides valuable information for the degree of bone marrow failure and transfusion necessity in cancer patients. 7,8 Furthermore, a more recent report has shown that sTfR level in urine potentially useful in diagnosis of IgA nephropathy (IgAN) and Henoch–Schönlein purpura nephritis (HSPN).9 In view of all these applications, the quantification of sTfR level is of great importance in clinical practice and biomedical studies. Most of the current methods for sTfR quantification rely on immunological methods, e. g. immunoturbidimetry, immunonephelometry, and enzyme-linked immunosorbent assays (ELISA). 10-13 These antibody-based methods are labor-intensive, not easy to scale up, and lack of reproducibility. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)–based targeted proteomics has been employed as an accurate alternative but it requires sophisticated instruments.7 Later, methods involving clinical analyzers offer automatic assays with high sample throughput, which exhibit superior precision over traditional ELISA assays, such as immunoturbidimetric assay based on Roche Hitachi analyzer.14,15 More recently, quantification of sTfR has been realized by molecular imprinting strategy and enhanced sandwich immunoassay integrated on a photonic crystal biosensor.16,17 However, most of these methods still require sophisticated instruments and professional personnel to operate. Fluorogens with aggregation-induced emission (AIE) characteristics have been widely applied in chemo/biological sensing during the past decades.18,19, 20 AIEgens hardly show fluorescence when molecularly dissolved in benign solvents, but they fluoresce strongly in the solid/aggregate state due to the restriction of intramolecular motion (RIM) effect.21,22 Taking advantage of this feature, light-up probes have been developed by conjugation of hydrophilic moieties with aromatic AIEgens, which show subsequent fluorescence turn-on upon analyte recognition. 23-25 The first generation of AIE probes was mainly designed based on 3 ACS Paragon Plus Environment


Page 4 of 16

electrostatic/hydrophobic interactions.26-30 Later, a diverse of AIE-probes are developed by introducing sugar,31,32 oligonucleotide,33,34 or peptide35-37 as hydrophilic recognition moieties. These probes facilitate real-time, rapid, sensitive, and specific detection of analytes, which have been successfully employed as sensing,31-35 imaging36-39 and therapeutic tools40,41. sTfR is a truncated monomer of transferrin receptor (TfR), lacking its first 100 amino acids.4 A matrix metalloproteinase cleaved between arginine-100 and leucine-101 of the extracellular domain of the TfR, releasing the sTfR into the serum. Considering the structural similarity of TfR and sTfR, we now applied the TfR-targeting peptide T7 (His-Ala-Ile-Tyr-Pro-Arg-His, HAIYPRH) for the sensing of sTfR. In this work, we have synthesized a green-emissive probe for specific light-up detection of sTfR. The probe consists of three components: (1) an AIEgen TPE showing fluorescence turn-on when molecularly restricted or upon aggregation formation but remaining nonemissive in aqueous media; (2) a peptide moiety T7; and (3) azide and alkyne groups functionalized on the TPE and peptide, respectively for further covalent conjugation. Experimental Section Synthesis of compound 2 (TPE-2I): To an acetone solution (50 mL) of compound 1 (250 mg, 0.69 mmol), potassium carbonate (286 mg, 2.07 mmol) and 1,4-diiodobutane (0.20 mL, 1.51 mmol) were added under ice-bath. The mixture was refluxed for 12 h, then acetone was evaporated by compressed air, and water and dichloromethane (DCM) were subsequence added for solvent extraction. The collected organic layer was dried over anhydrous magnesium sulfate after washing with brine. The resulting extract was evaporated under reduced pressure and subjected to column chromatography with ethyl acetate and hexane (1:9, v/v) as eluent to afford compound 2 TPE-2I as a pale-yellow oillike compound (229 mg, 45.8%).1H NMR (400 MHz, CDCl3): δ (TMS, ppm): 7.10-7.04 (10H, m), 7.01-6.99 (4H, d), 6.91-6.89 (4H, d), 3.89 (4H, t), 3.23 (4H, t), 2.03-1.8 (8H, m). Mass spectrum (MALDI-TOF), m/z calcd. for C34H34I2O2: 728.0648, found: 728.0657 [M]+. Synthesis of compound 3 1,1-bis[4-(azidobutoxy)phenyl]-1,1-diphenylethene (TPE-2N3): A mixture of compound 2 (229 mg, 0.314 mmol) and sodium azide (72 mg, 1.10 mmol) in dimethyl sulfoxide (20 mL) was stirred at room temperature overnight. Diethyl ether and water were poured into the reaction mixture. The combined organic layer was washed with brine and dried over anhydrous magnesium sulfate and evaporated to dryness. The desirable product was purified by column chromatography with ethyl acetate and hexane (1:9, v/v) as eluent to afford compound 3 as a pale-yellow oil-like compound (127 mg, 36.5%). 1H NMR (400 MHz, CDCl3): δ (TMS, ppm) 7.10-6.60 (18H, m), 3.91 (4H, t), 3.34 (4H, t), 1.82-1.76 (8H, m). 13C NMR (400 MHz, CDCl3): δ (TMS, ppm) 157.1, 144.0, 139.5, 138.9, 136.2, 132.4, 131.1, 127.4, 125.8, 113.2, 66.7, 4 ACS Paragon Plus Environment

Page 5 of 16 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


51.0, 26.3, 25.5. Mass spectrum (MALDI-TOF), m/z calcd. for C34H34N6O2: 558.2743, found: 558.2767 [M]+. Synthesis of the probe TPE-2T7 TPE-2N3 (3.0 mg, 5.37 μmol) and alkyne-T7 peptide (Alkyne-HAIYPRH, 13.3 mg, 12.89 μmol) was dissolved in the mixture of DMSO and water (v/v = 1:1). Sodium ascorbate (3.7 mg, 18.80 μmol) copper sulfate (2.1 mg, 12.89 μmol) were added into the mixture to catalyze the click reaction. The mixture was then allowed to further reacted for 24 h The final product was purified by HPLC and lyophilized to obtain the pure product as a white solid in 85% yield. The structure and purity of the product was characterized by 1H NMR and HRMS. 1H NMR (400 MHz, DMSO-d6), δ (TMS, ppm): 8.98-8.97 (m, 4H), 8.31-8.29 (d, J = 8.0 Hz, 2H), 8.18-8.09 (m, 10 H), 7.92-7.90 (d, J = 8.0 Hz, 2H), 7.82 (s, 2H), 7.65-7.62 (t, J = 5.6 Hz, 2H), 7.35 (s, 5H), 7.26 (s, 2H), 7.15-7.04 (m, 11H), 6.96-6.94 (m, 5H), 6.86-6.84 (d, d, J = 8.0 Hz, 4H), 6.68-6.61 (m, 8H), 4.66-4.41 (m, 8H), 4.38-4.30 (m, 8H), 4.20-4.15 (m, 5H), 3.66-3.65 (m, 2H), 3.48-3.47 (m, 2H), 3.17-3.09 (m, 8H), 3.03-2.94 (m, 6H), 2.87-2.81 (m, 4H), 2.74-2.63 (m, 2H), 2.03-1.99 (m, 2H), 1.94-1.87 (m, 6H), 1.84-1.80 (m, 9H), 1.68-1.55 (m, 7H), 1.51-1.44 (m, 5H), 1.35-1.30 (m, 2H), 1.28-1.23 (m, 2H), 1.20-1.18 (d, J = 8.0 Hz, 5H), 1.03-0.96 (m, 2H), 0.78-0.72 (m, 12H). HRMS (ESI-MS): m/4z [M+4H]4+ calcd for C130H174N38O22: 654.8411; found: 654.8404. Synthesis of graphene oxide (GO) Graphene oxide (GO) was synthesized according to a modified Hummers method.42,43 Specifically, into the mixture of 500 mg graphite powder, 700 mg NaNO3 and 3.1 g KMnO4, 35 mL sulfuric acid was added dropwisely with ice-bath. The mixture was stirred at 35oC for 1.5 hours, followed by the addition of 40 mL water. After further stirring at 95 oC for 1.5 hours, another 100 mL water was gradually added. The mixture was further oxidized by addition of 3.6 mL H2O2 (36 %), turning the color of reaction liquid from dark brown to yellow. After repeated wash and dialysis to remove acid, graphene oxide was obtained. The concentration was determined by lyophilization of a certain volume of the GO solution. The concentration of GO used in this work is 1.0 mg/mL. Procedure for light-up detection of sTfR using probe TPE-2T7 In this work, all the titration experiments were carried out in PBS buffers containing 13.7 mM NaCl, 0.27 mM KCl, 0.8 mM Na2HPO4, and 0.2 mM KH2PO4. Firstly, 5 mM TPE-2T7 stock solution in DMSO was diluted to 10 µM in DMSO/PBS buffer (v/v = 1/99). Then an increasing amount of recombinant sTfR protein was added into the probe solution. The PL spectra of the probe solution in the presence of 0, 5, 10, 20, 40, 60, 80, and 100 µg/mL sTfR were collected from 400 – 600 nm with excitation at 315 nm. The selectivity of the probe was evaluated by incubation



Page 6 of 16

with 100 μg/mL of other proteins including pepsin, lysozyme, human serum albumin (HSA), transferrin (Tf), and the mixture of them in the same mixture solvent DMSO/PBS (v/v = 1/99). Light-up detection of sTfR in urine samples Urine samples were collected from normal people and labelled properly. Twenty 5-mL urine aliquots were used as control samples from normal people. Ten 5-mL urine aliquots were spiked with sTfR at a final concentration of 5-30 µg/L. All the urine samples were pretreated by filtration using centrifugal filters with a cutoff molecular weight of 10 kDa to remove interferants at low molecular weight range. All the urine samples were then concentrated 50fold before usage. The thirty urine samples were then labeled randomly and used for sTfR detection experiments. For the detection experiments, TPE-2T7 stock solution in DMSO was added to each urine samples to obtain the probe solution at the concentration of 10 μM. To further eliminate the background signal and increase the S/N ratio, different amount of 1 mg/mL GO solution was added to 1 mL of the probe solution. Then the PL intensity of the samples were collected and recorded in the presence with proper concentration of GO. Cell culture To investigate the potential of the probe TPE-2T7 for imaging of transferrin receptor (TfR) and discrimination of TfR-overexpressed cancer cells over normal cells, MDA-MB-231 human breast cancer cells and NIH 3T3 cells were chosen as the TfR positive and negative model. All the cells were cultured in DMEM purchased from Invitrogen containing 10% FBS and 1% penicillin streptomycin at 37oC in a humidified environment (5% CO2). Before experiments, the cells were precultured to reach confluence. Results and Discussion Probe design and synthesis The probe consists of three elements, a green-emissive AIEgen TPE as the reporter moiety, peptide T7 (HAIYPRH) moiety allowing specific binding with sTfR, and alkyne and azide groups facilitating the covalent conjugation between the reporting and targeting groups. As shown in Scheme 1, Compound 1 was synthesized according to previous literature in 93.4% yield.41,44 Then compound 1 was reacted with 1,4-diiodobutane in the presence of potassium carbonate in acetone solution to yield compound 2 after purification by column chromatography in 45.8% yield. Compound 2 was further reacted with sodium azide to yield compound 3 1,1-bis[4(azidobutoxy)phenyl]-1,1-diphenylethene in 36.5% yield. Finally, the probe TPE-2T7 was obtained in 50% yield via the click chemistry between compound 3 and the alkyne functionalized peptide HAIYPRH. The chemical structures of TPE-2N3 and the probe TPE-2T7 were confirmed by 1H NMR and HRMS with high purity, as shown in Figures S1-S5 in the 6 ACS Paragon Plus Environment

Page 7 of 16 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


supporting Information, respectively. The probe is expected to show very weak emission in aqueous media but can fluoresce strongly when binding with sTfR.

Scheme 1 Synthetic Routes to TPE-2N3 and the probe TPE-2T7. Optical Properties The UV-vis absorption and photoluminescence (PL) spectra of TPE-2N3 and TPE-2T7 were measured in the mixture of DMSO/H2O (v/v = 1/99) at the concentration of 10 μM. Figure 1A shows that TPE-2N3 has an absorption peak at 325 nm, which is blue-shifted to 315 nm after conjugation with T7 peptide. As expected, TPE-2N3 is very emissive in aqueous media but it becomes almost non-emissive once it is conjugated with T7 peptide (Insets in Figure 1A). As shown in the laser light scattering (LLS) results, TPE-2N3 stays as aggregates in aqueous media with hydrodynamic diameter of 100 ± 10 nm (Figure S6). In contrast, no LLS signal was observed for TPE-2T7, indicating that the probe has completely dissolved in the aqueous media due to its good water-solubility. To further examine the AIE characteristics, the PL spectra of TPE-2N3 were measured in the mixed solvents of DMSO/H2O with increasing water fractions and the results are shown in Figure S7. The PL intensities at the emission maxima are shown in Figure 1B. TPE-2N3 remains nonemissive when the water fractions stay below 40%, which is followed by a dramatic increase when water fraction increases from 40% to 50%. The peak PL intensities 7 ACS Paragon Plus Environment


Page 8 of 16

of TPE-2N3 increase gradually when water fractions ranging from 50% to 99%, which is due to the aggregates formation of aromatic TPE-2N3 in aqueous media. In contrast, the probe TPE2T7 only shows very faint fluorescence even in the mixture of DMSO/H2O (v/v = 1/99). The good solubility of TPE-2T7 endows it low fluorescence, which is desirable for the light-up sensing of sTfR.

Figure 1 (A) UV-vis absorption and photoluminescence (PL) spectra of TPE-2N3 and TPE-2T7 in DMSO/PBS buffer (v/v = 1/99); (B) plots of emission maxima of TPE-2N3 in DMSO/H2O mixture with increasing water fractions. λex (TPE-2N3) = 325 nm; λex (TPE-2T7) = 315 nm; [TPE-2N3] = [TPE-2T7] = 10 μM. Light-up detection of sTfR using probe TPE-2T7 To validate the potential of probe TPE-2T7 for light-up detection of sTfR, its PL intensity was measured in the presence of sTfR with different concentrations. Figure 2A shows that 10 μM TPE-2T7 in the DMSO/PBS buffer is only very weekly emissive but its fluorescence turns on upon addition of sTfR. The PL spectra of the probe TPE-2T7 incubated with 0, 5, 10, 20, 40, 60, 80, and 100 µg/mL sTfR are shown in Figure 2A. The plots of PL intensity at 480 nm against the increasing concentration of sTfR are summarized in Figure 2B. As shown, there is a linear trend in the range of 0-80 µg/mL with a R2 = 0.99. The detection limit was calculated to be 0.27 µg/mL by (I0 + 3SD), where I0 and SD represent the fluorescence intensity and the standard deviation of blank sample. The normal concentration of sTfR is about 1.0-2.9 µg/mL in adults, which may increase up to 20-fold when there is iron deficiency.45 It is validated that the probe TPE-2T7 provides linearly turn-on fluorescent signal upon interaction with the increasing amount of target sTfR. The rationale behind this fluorescence light-up can be attributed to the restriction of intramolecular motion of TPE upon binding to protein sTfR. We further measured the zeta potential of the probe before and after binding with sTfR. The zeta potential was measured to be 18.5 ± 8.06 mV (before binding) and -0.3 ± 3.78 mV (after binding), respectively, which are in accordance with the charge property of TPE-2T7 and sTfR, respectively (as shown in Figure S8). 8 ACS Paragon Plus Environment

Page 9 of 16

In view of the above results, it could be concluded that the peptide sequence T7 binds with the protein sTfR. 0 5 10 20 40 60 80 100 µg / mL sTfR

A 150 120 90 60

B 150 120 90 60 30

30 0 400

180

PL Intensity (a.u.)

180

PL Intensity (a.u.)

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


y = 1.63x + 3.25

0

450

500

550

Wavelength (nm)

600

0

25

50

75

100

sTfR (µg / mL)

Figure 2 (A) photoluminescence (PL) spectra of TPE-2T7 in the presence of increasing amount of sTfR at the concentration of 0, 5, 10, 20, 40, 60, 80, and 100 µg/mL in DMSO/PBS buffer (v/v = 1/99). (B) Plots of PL intensity at 475 nm of TPE-2T7 against the increasing concentration of sTfR. [TPE-2T7] = 10 μM, error bars represent standard deviation, n = 3. The selectivity of TPE-2T7 was further examined by incubation of the probe with other proteins. Specifically, 10 μM TPE-2T7 in DMSO/PBS buﬀer (v/v = 1/99) was incubated with proteins including sTfR, pepsin, human serum albumin (HSA), lysozyme, transferrin (Tf), and sTfR with all the proteins mixed together. Pepsin, HSA, and lysozyme have isoelectric points (pI) of 2.9, 4.7, and 11.35, respectively, which show negative, negative and positive charges under pH 7.4. Transferrin (Tf) has also been taken into consideration because the circulation of sTfR is usually accompanied by Tf. The PL intensity of each mixture is shown in Figure 3. As expected, the probe TPE-2T7 only shows fluorescence turn-on in the presence of sTfR but remains nonemissive when it is incubated with other proteins. However, the fluorescence of the probe can be turned on by the sTfR when it is mixed with other proteins. The above results prove that the fluorescence enhancement of the probe TPE-2T7 is due to its specific interaction with sTfR, which is not affected by the presence of other proteins.



80

60

(I-I0 )/I0

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

Page 10 of 16

40

20

0 e in fR sT Peps ozym Lys

Tf

A HS fR sT

+

er oth

n tei p ro

Figure 3. PL enhancement upon incubation of the probe TPE-2T7 with sTfR and other proteins including pepsin, lysozyme, human serum albumin (HSA), transferrin (Tf), and the mixture of these proteins. [protein] = 100 μg/mL, [TPE-2T7] = 10 μM. Proof-of-concept detection of sTfR in urine sample Although the detection limit of the probe TPE-2T7 is far below the reported sTfR level in the serum of IDA patients, there are still some issues before the probe could be applied to the detection of sTfR in human serum. In every milliliter of serum, there are 60-80 mg of various proteins. Among these proteins, a few high-abundant ones constitute 90% of the total serum content, while the rest 10% consists of a variety of low-abundant proteins. The abundant proteins significantly affect the sensitivity and selectivity of many techniques, which hinders the detection of low-abundant proteins, let alone the disease markers at trace amount such as sTfR. Hence, the most challenging part for serum-related detection is sample preparation. For instance, depletion process is required to remove high-abundant proteins in serum. In addition, protein/peptide immunoaffinity enrichment has been utilized to improve the detection sensitivity.7 Alternatively, urine tests can provide more accurate clinical information. Urine has become an important biomarker source for diagnosis of bladder cancer, prostate cancer, ovarian cancer and kidney diseases. Compared with blood samples, urine samples are easier to handle due to their less complexity in monitoring the changes of low-abundant proteins.46 As stated above, the TfR/sTfR reflect totally different physiological processes when they are at different locations in human bodies. In this section, we utilize the probe TPE-2T7 in the detection of sTfR in urine samples as a proof-of-concept for the diagnosis of IgA nephropathy (IgAN) and Henoch– Schönlein purpura nephritis (HSPN).


Page 11 of 16

To validate the potential of the probe TPE-2T7 for the detection sTfR in urine, 20 artificial patient samples spiked with sTfR were labeled randomly as the analytes for sTfR detection. 10 normal samples were prepared for determination of background signals from the blank samples. In addition to amplifying detection signal, another way to improve the overall sensitivity is to lower the background signal. Graphene oxide (GO) is a good option as a quencher to eliminate the background fluorescence of probes.47 As shown in the Figure S9, 2.5 μg/mL GO significantly quench the fluorescence of the probe TPE-2T7 in the aqueous media. Then the PL intensity of all the samples were measured after addition of 10 μM TPE-2T7 in the presence of the same concentration of GO. As shown in Figure 4, all the “patient samples” can induce decent fluorescence enhancement above the threshold of (I0 + 3σ). (I0 represents average fluorescence intensity at 480 nm of 10 blank samples; σ stands for standard deviation of 5 blank samples). The results show that 100% (20 out of 20) patient samples can be determined by our probe. The experiments validate the usefulness of the probe TPE-2T7 for the detection of sTfR in urine sample which shows great potential to be used for the diagnosis of kidney disease such as HSPN. 600

PL Intensity @ 480 nm

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


500 400 300 200 100 0 0

5

10

15

20

Samples entry

Figure 4 Detection of sTfR in urine samples by measurement of PL intensities at 480 nm from the probe TPE-2T7 in the presence of 2.5 μg/mL GO. The dash line represents the threshold of (I0 + 3σ). Error bars indicate standard deviation of triplicate tests. Targeted imaging of overexpressed TfR in cancer cells. The sTfR is a truncated monomer of transferrin receptor (TfR), lacking its first 100 amino acids. sTfR circulating in serum is closely related to the total amount of cell-associated TfR.48 However, TfR and sTfR exist in different locations and have different physiological significance. It would be great if a probe can detect both sTfR and TfR, which is meaningful for the further investigation of sTfR and TfR related questions. The peptide T7 has been applied for the targeting of TfR.2,49 To test whether the probe TPE-2T7 can detect TfR overexpressed in cancer cells, cell experiments were carried out using MDA-MB-231 and NIH 3T3 cells as the TfR11 ACS Paragon Plus Environment


Page 12 of 16

positive and negative cells. After incubation with the probe, as shown in Figure 5, TfRoverexpressed MDA-MB-231 cells show bright fluorescence. In contrast, the NIH 3T3 cells are almost nonemissive. These results indicate the probe TPE-2T7 can realize targeted imaging of TfR-overexpressed cancer cells.

Figure 5 CLSM images of the probe TPE-2T7 (10 µM) incubated with (A) MDA-MB-231 and (B) NIH 3T3 cells for 0.5 h. The images were taken with excitations at 405 nm (filter band passes: 430 - 560 nm.)

Conclusion The sTfR level in serum isa key parameter in diagnosis of iron deficiency anemia (IDA). In addition, its level in urine is closely related with prostate cancer and kidney cancer. In this work, we have developed a green-emissive AIE-peptide conjugate TPE-2T7 for fluorescent light-up detection of sTfR. The probe itself is almost nonemissive in aqueous solution, but it turns on its fluorescence upon interaction with sTfR to yield a detection limit of 0.27 µg/mL, which is much lower than the sTfR level in IDA patients. Furthermore, a proof-of-concept experiment validates the potential of the probe for diagnosis of HSPN by artificial urine test. sTfR and TfR share considerable structural similarity, but sTfR circulates in serum while TfR is a transmembrane protein. They have different physiological significance. The CLSM images reveal that the probe TPE-2T7 can also light-up on the cancer cell membrane upon specific binding with overexpressed TfR.

ASSOCIATED CONTENT Supporting Information 12 ACS Paragon Plus Environment

Page 13 of 16 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


Synthesis details and characterization (1H NMR, 13C NMR, and HRMS) of TPE-2N3 and the probe TPE-2T7; PL spectra of TPE-2N3 in the mixture of DMSO/H2O with different water fractions, laser light scattering resutls of TPE-2N3, Zeta potential of the probe TPE-2T7 before and after binding with sTfR; These material is available free of charge via the Internet at http://pubs.acs.org.” For instructions on what should be included in the Supporting Information, as well as how to prepare this material for publication, refer to the journal’s Instructions for Authors. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]; [email protected] Author Contributions R. Zhang and Simon H. P. Sung contributed equally to this work. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT We thank the National University of Singapore (R279-000-482-133), Singapore NRF Investigatorship (R279-000-444-281), National Research Foundation (R279-000-483-281) and Innovation and Technology Commission (ITC-CNERC14SC01) for financial support.

REFERENCES (1) Daniels, T. R.; Delgado, T.; Helguera, G.; Penichet, M. L. Clin. Immunol. 2006, 121, 159-176. (2) Han, L.; Guo, Y.; Ma, H.; He, X.; Kuang, Y.; Zhang, N.; Lim, E.; Zhou, W.; Jiang, C. Small 2013, 9, 36473658. (3) Cao, C.; Wang, X.; Cai, Y.; Sun, L.; Tian, L.; Wu, H.; He, X.; Lei, H.; Liu, W.; Chen, G. Adv. Mater. 2014, 26, 2566-2571. (4) Cook, J.; Skikne, B.; Baynes, R. Annu. Rev. Med. 1993, 44, 63-74. (5) Baillie, F.; Morrison, A.; Fergus, I. Clin. Lab. Haematology 2003, 25, 353-357. (6) Pettersson, T.; Kivivuori, S.-M.; Siimes, M. A. Br. J. Rheumatol. 1994, 33, 740-744. (7) Xu, Q.; Zhu, M.; Yang, T.; Xu, F.; Liu, Y.; Chen, Y. Clin. Chim. Acta 2015, 448, 118-123. (8) Dowlati, A.; Loo, M.; Bury, T.; Fillet, G.; Beguin, Y. Br. J. Cancer 1997, 75, 1802. (9) Delanghe, S. E.; Speeckaert, M. M.; Segers, H.; Desmet, K.; Vande Walle, J.; Laecke, S. V.; Vanholder, R.; Delanghe, J. R. Clin. Biochem. 2013, 46, 591-597. (10) Erhardt, J. G.; Estes, J. E.; Pfeiffer, C. M.; Biesalski, H. K.; Craft, N. E. J. Nutr. 2004, 134, 3127-3132. (11) Infusino, I.; Braga, F.; Dolci, A.; Panteghini, M. Am. J. Clin. Pathol. 2012, 138, 642-649. (12) Abellan, R.; Ventura, R.; Pichini, S.; Sarda, M.; Remacha, A.; Pascual, J.; Palmi, I.; Bacosi, A.; Pacifici, R.; Zuccaro, P. J. Immunol. Methods 2004, 295, 89-99. 13 ACS Paragon Plus Environment


Page 14 of 16

(13) Hikawa, A.; Nomata, Y.; Suzuki, T.; Ozasa, H.; Yamada, O. Clin. Chim. Acta 1996, 254, 159-172. (14) Pfeiffer, C. M.; Cook, J. D.; Mei, Z.; Cogswell, M. E.; Looker, A. C.; Lacher, D. A. Clin. Chim. Acta 2007, 382, 112-116. (15) Kolbe-Busch, S.; Lotz, J.; Hafner, G.; Blanckaert, N. J.; Claeys, G.; Togni, G.; Carlsen, J.; Röddiger, R.; Thomas, L. Clin. Chem. Lab. Med. 2002, 40, 529-536. (16) Peterson, R. D.; Chen, W.; Cunningham, B. T.; Andrade, J. E. Biosens. Bioelectron. 2015, 74, 815-822. (17) Liu, L.; Zhong, T.; Xu, Q.; Chen, Y. Anal. Chem. 2015, 87, 10910-10919. (18) Mei, J.; Leung, N. L.; Kwok, R. T.; Lam, J. W.; Tang, B. Z. Chem. Rev. 2015, 115, 11718-11940. (19) Hong, Y.; Lam, J. W.; Tang, B. Z. Chem. Commun. 2009, 4332-4353. (20) Hong, Y.; Lam, J. W.; Tang, B. Z. Chem. Soc. Rev. 2011, 40, 5361-5388. (21) Parrott, E. P.; Tan, N. Y.; Hu, R.; Zeitler, J. A.; Tang, B. Z.; Pickwell-MacPherson, E. Mater. Horiz. 2014, 1, 251-258. (22) Mei, J.; Hong, Y.; Lam, J. W.; Qin, A.; Tang, Y.; Tang, B. Z. Adv. Mater. 2014, 26, 5429-5479. (23) Liang, J.; Tang, B. Z.; Liu, B. Chem. Soc. Rev. 2015, 44, 2798-2811. (24) Ding, D.; Li, K.; Liu, B.; Tang, B. Z. Acc. Chem. Res. 2013, 46, 2441-2453. (25) Kwok, R. T.; Leung, C. W.; Lam, J. W.; Tang, B. Z. Chem. Soc. Rev. 2015, 44, 4228-4238. (26) Hong, Y.; Meng, L.; Chen, S.; Leung, C. W. T.; Da, L.-T.; Faisal, M.; Silva, D.-A.; Liu, J.; Lam, J. W. Y.; Huang, X. J. Am. Chem. Soc. 2012, 134, 1680-1689. (27) Hong, Y.; Feng, C.; Yu, Y.; Liu, J.; Lam, J. W. Y.; Luo, K. Q.; Tang, B. Z. Anal. Chem. 2010, 82, 70357043. (28) Wang, M.; Zhang, D.; Zhang, G.; Tang, Y.; Wang, S.; Zhu, D. Anal. Chem. 2008, 80, 6443-6448. (29) Zhuang, Y.; Zhang, M.; Chen, B.; Duan, R.; Min, X.; Zhang, Z.; Zheng, F.; Liang, H.; Zhao, Z.; Lou, X.; Xia, F. Anal. Chem. 2015, 87, 9487-9493. (30) Wang, M.; Gu, X.; Zhang, G.; Zhang, D.; Zhu, D. Anal. Chem. 2009, 81, 4444-4449. (31) Sanji, T.; Shiraishi, K.; Nakamura, M.; Tanaka, M. Chem. –Asian J. 2010, 5, 817. (32) Sanji, T.; Shiraishi, K.; Tanaka, M. ACS Appl. Mater. Interfaces 2008, 1, 270-273. (33) Min, X.; Zhuang, Y.; Zhang, Z.; Jia, Y.; Hakeem, A.; Zheng, F.; Cheng, Y.; Tang, B. Z.; Lou, X.; Xia, F. ACS Appl. Mater. Interfaces 2015, 7, 16813-16818. (34) Li, Y.; Kwok, R. T.; Tang, B. Z.; Liu, B. RSC Adv 2013, 3, 10135-10138. (35) Shi, H.; Liu, J.; Geng, J.; Tang, B. Z.; Liu, B. J. Am. Chem. Soc. 2012, 134, 9569-9572. (36) Shi, H.; Kwok, R. T. K.; Liu, J.; Xing, B.; Tang, B. Z.; Liu, B. J. Am. Chem. Soc. 2012, 134, 17972-17981. (37) Wang, Y.; Chen, Y.; Wang, H.; Cheng, Y.; Zhao, X. Anal. Chem. 2015, 87, 5046-5049. (38) Yuan, Y.; Zhang, C.-J.; Xu, S.; Liu, B. Chem. Sci. 2016, 7, 1862-1866. (39) Yuan, Y.; Zhang, C.-J.; Kwok, R. T. K.; Xu, S.; Zhang, R.; Wu, J.; Tang, B. Z.; Liu, B. Adv. Funct. Mater. 2015, 25, 6586-6595. (40) Yuan, Y.; Kwok, R. T.; Tang, B. Z.; Liu, B. J. Am. Chem. Soc. 2014, 136, 2546-2554. (41) Yuan, Y.; Zhang, C. J.; Gao, M.; Zhang, R.; Tang, B. Z.; Liu, B. Angew. Chem. Int. Ed. 2015, 54, 17801786. (42) William, S.; Hummers, J.; Offeman, R. J. Am. Chem. Soc. 1958, 80, 1339. (43) Marcano, D. C.; Kosynkin, D. V.; Berlin, J. M.; Sinitskii, A.; Sun, Z.; Slesarev, A.; Alemany, L. B.; Lu, W.; Tour, J. M. ACS Nano 2010, 4, 4806-4814. (44) Hu, R.; Lam, J. W.; Yu, Y.; Sung, H. H.; Williams, I. D.; Yuen, M. M.; Tang, B. Z. Polym. Chem. 2013, 4, 95-105. (45) Khatami, S.; Dehnabeh, S. R.; Mostafavi, E.; Kamalzadeh, N.; Yaghmaei, P.; Saeedi, P.; Shariat, F.; Bagheriyan, H.; Zeinali, S.; Akbari, M. T. Hemoglobin 2013, 37, 387-395. (46) Shao, C.; Wang, Y.; Gao, Y. Sci. China: Life Sci. 2011, 54, 409-417. (47) Chung, C.; Kim, Y.-K.; Shin, D.; Ryoo, S.-R.; Hong, B. H.; Min, D.-H. Acc. Chem. Res. 2013, 46, 22112224. 14 ACS Paragon Plus Environment

Page 15 of 16 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


(48) Allen, J.; Backstrom, K. R.; Cooper, J. A.; Cooper, M. C.; Detwiler, T. C.; Essex, D. W.; Fritz, R. P.; Means, R. T.; Meier, P. B.; Pearlman, S. R. Clin. Chem. 1998, 44, 35-39. (49) Zhang, R.; Feng, G.; Zhang, C.-J.; Cai, X.; Cheng, X.; Liu, B. Anal. Chem. 2016, 88, 4841-4848.



Page 16 of 16

Table of Content


Aggregation-Induced Emission Probe for Specific Turn-On

Recommend Documents