Recombinant Protein (Luciferase-IgG Binding Domain) Conjugated

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Recombinant Protein (luciferase-IgG binding domain) Conjugated Quantum Dots for BRET-Coupled Nearinfrared Imaging of Epidermal Growth Factor Receptors Takashi Jin, and Setsuko Tsuboi Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00149 • Publication Date (Web): 08 Mar 2018 Downloaded from http://pubs.acs.org on March 8, 2018

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Bioconjugate Chemistry

Recombinant Protein (luciferase-IgG binding domain) Conjugated Quantum Dots for BRET-Coupled Near-infrared Imaging of Epidermal Growth Factor Receptors Setsuko Tsuboia and Takashi Jina,b * a

Quantitative Biology Center, RIKEN, Furuedai 6-2-3, Suita, Osaka 565-0874, Japan

b

Graduate School of Frontier Biosciences, Osaka University, Yamada-oka 2-1, Suita, Osaka

565-0871, Japan

KEYWORDS: Near-infrared (NIR); bioluminescence; bioluminescence resonance energy transfer (BRET); quantum dot (QD); luiciferase; immunogloblin G (IgG); epidermal growth factor receptor (EGFR).

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ABSTRACT：For the highly sensitive near-infrared (NIR) optical detection of epidermal growth factor receptors (EGFRs) expressed on cancer cells, bioluminescence resonance energy transfer (BRET) coupled NIR quantum dots (QDs) are prepared by direct conjugation of his-tagged Renilla luciferase (RLuc) recombinant protein (HisRLuc·GB1) to glutathione coated CdSeTe/CdS QDs (GSH-QDs). The recombinant protein has two functional groups consisting of a luciferase enzyme and an immunoglobulin binding domain (GB1) of protein G. Recombinant protein (HisRLuc·GB1) conjugated QDs (GB1·RLuc-QDs) show BRETcoupled NIR emission, which results from energy transfer from luciferin to QDs with a high BRET efficiency of ca. 50 %. Since the GB1·RLuc-QDs have the GB1 domain at their surface, the QDs have an ability to bind the Fc moiety of immunoglobulin G (IgG). The resulting IgG bound QDs can be used as a molecular imaging probe with NIR fluorescence and BRET-coupled NIR emission. For NIR optical detection of EGFRs on cancer cells, we conjugated anti-EGFR monoclonal antibody to the GB1·RLuc-QDs. Herein, we show that the detection sensitivity of EGFRs by BRET-coupled NIR emission of GB1·RLuc-QDs is at least three times higher than that of the NIR fluorescence of the QDs. The conjugates between anti-EGFR antibody and GB1·RLuc-QDs make it possible to perform BRET-based highly sensitive NIR imaging of EGFRs in living cells.


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INTRODUCTION Bioluminescence imaging using the luciferase-luciferin system is widely used for biosensing and bioimaging in life science studies.1,2 In the bioluminescence imaging, external excitation is not needed, leading to the low background signal (e.g. autofluorescence) which results from numerous fluorescent species in biological samples.3,4 Thus, the bioluminescence imaging is expected to offer highly sensitive imaging with wider dynamic range of signals compared with fluorescence imaging.2 Many bioluminescent species existing in nature emit in the visible region. For instance, firefly luciferase-D-luciferin which emits at around 560 nm is the most widely

used

bioluminescence

system

in

biomedical

research.1

The

visible

bioluminescence has a disadvantage that the luminescence is strongly absorbed by intrinsic chromophores such as hemoglobin and melanin in tissues.5 For the improvement of signal to background ratios in the bioluminescence imaging, bioluminescence with longer wavelengths beyond visible region is desirable. To date, luciferin analogues that generate bioluminescent emissions in NIR region have been synthesized.6-10 Several groups have reported luciferin derivatives with extended pconjugation for NIR bioluminescence imaging with longer wavelengths.6,8,10 Alternatively, BRET-coupled emission from luciferin to NIR fluorophores has been employed for bioluminescence imaging in NIR wavelength regions, where organic dyes,7,11,12 fluorescent proteins13,14 and quantum dots (QDs)/nanoparticles15-35 are used as energy acceptors. Rao et al. have reported BRET-FRET-coupled NIR nanoprobes for in vivo lymphnode mapping and tumour imaging.29 They conjugated luciferase proteins to NIR-dye containing nanoparticles via carbodiimide coupling reaction between amino groups of luciferase and carboxyl groups presented on the surface of the nanoparticles. Recently, Cai et al. have reported BRET-coupled NIR emission based on Nano luciferase (Oplophorus


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gracilirostris)-QD conjugates for in vivo lymph-node mapping and tumor imaging,34 where the carbodiimide coupling is also used for the conjugation of the Nano luciferase to NIR emitting QDs. Although several types of BRET-coupled fluorophores have been developed as imaging/sensing probes, 15-35 there are a very few reports on molecular targeting probes based on BRET-coupled NIR emission.26,29,34 For tumor imaging, cyclic arginine-glycineaspartic acid (RGD) peptides as the targeting ligands have been conjugated to the luciferaseQD conjugates.29,34 Stephan et al. developed an ultrasensitive method for the detection of cellular proteins using BRET-coupled emission from antibody conjugates with luciferase labeled QDs.26 They achieved molecular imaging of human epidermal growth factor 2 (HER2) receptors on cell membrane using luciferase conjugated QDs, where the QD surface was modified with a biotinylated anti-HER2 antibody. To develop QD-based molecular probes, QD surface is usually modified with antibodies, peptides, carbohydrates, and receptor ligands.36,37 Among the surface-functionalized QDs, antibody conjugated QDs are the most promising probes for molecular imaging.38-40 In this paper, we present a facile method for the preparation of antibody conjugates with BRETcoupled NIR-QDs as a molecular targeting probe for epidermal growth factor receptors (EGFRs)41,42 on cancer cells. EGFR is a transmembrane glycoprotein, which is overexpressed in a variety of solid tumours such as breast, lung, and gastric cancer. BRET-coupled NIRQDs are prepared by direct conjugation of a recombinant Renilla luciferase (RLuc) protein (HisRLuc·GB1) to the surface of gultathione coated QDs (GSH-QDs)43,44 via N-terminus hexahistidine tags (His6) of the recombinant protein. Since the recombinant protein include an immunoglobulin binding domain45 (GB1) of protein G, resulting BRET coupled QDs (GB1·RLuc-QDs) can bind the Fc moiety of antibody (immunoglobulin G, IgG) at their surface. Herein, we show that BRET-coupled NIR emission in the GB1·RLuc-QDs can be used for highly sensitive imaging of EGFRs in living cells. The BRET-coupled NIR emission


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of GB1·RLuc-QDs shows a high BRET efficiency (ca. 50 %), and the detection sensitivity of EGFRs by BRET-coupled NIR emission of the GB1·RLuc-QDs is at least three times higher than that by NIR fluorescence of the QDs.

RESULTS AND DISCUSSION Synthesis of BRET-coupled NIR-QDs (GB1·RLuc-QDs). BRET-coupled NIR-QDs (GB1·RLuc-QDs) was prepared from a his-tagged recombinant protein (HisRLuc·GB1) and glutathione-capped NIR-emitting CdSeTe/CdS QDs (GSH-QDs). In the BRET-coupled NIRQDs, HisRLuc·GB1 is directly conjugated to the surface of GSH-QDs only by mixing the his-tagged protein and QDs (Figure1). The HisRLuc·GB1 protein includes an immunoglobulin binding domain45 (GB1) of protein G. Thus, GB1·RLuc-QDs can bind antibody (immunoglobulin G, IgG) via its Fc moiety (Figure 1). Since the luminescence spectrum of CTZ overlaps with the absorption spectrum of GSH-QDs, it is expected that the BRET from a luciferase substrate, coelenterazine (CTZ) to GSH-QDs will occur. The GSHQDs show a very broad absorption spectrum ranging from the visible to NIR region, and they

Figure 1. Synthetic method for the preparation of BRET-coupled NIR-QDs (GB1·RLucQDs) from glutathione-capped QDs (GSH-QDs), and schematic representation for the BRET-coupled NIR emission from antibody conjugates with GB1·RLuc-QDs in the presence of coelenterazine (CTZ).


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emit at ca. 830 nm (Figure 2a). Transmission electron microscopy (TEM) image of the GSHQDs is shown in the inset of Figure 2a. A mean diameter of the GSH-QDs was determined to be 5.9 nm from the particle size distribution of QDs (Figure S1). The purity of HisRLuc·GB1 and HisRLuc (control) protein was checked by SDS polyacrylamide gel electrophoresis (Figure 2b).

Figure 2. (a) Fluorescence (solid line) and absorption (dotted line) spectrum of GSHQDs in aqueous solution. Bioluminescence spectrum of CTZ is shown as a blue dotted line. The inset shows a TEM image of the GSH-QDs. Scale bar, 10 nm. (b) SDS polyacrylamide gel electrophoresis of HisRLuc and HisRLuc·GB1. Characterization of BRET-coupled NIR-QDs (GB1·RLuc-QDs). The binding of HisRLuc·GB1 protein to GSH-QDs was confirmed by agarose gel electrophoresis and sizeexclusion HPLC (Figure 3a and 3b). The mobility of a NIR-emitting band of GSH-QDs decreased with increasing the amounts of HisRLuc·GB1 (Figure 3a), showing that the HisRLuc·GB1 protein binds to the surface of GSH-QDs via histidine tags (His6). Since histidine has complexing abilities toward divalent cations such as Ni2+, Zn2+, and Cd2+,46,47 his-tagged proteins are useful for labeling of proteins on the surface of QDs covering with Zn2+ and Cd2+ ions.35,48 Maye et al. have found that his-tagged proteins can be directly conjugated to the surface of CdSe/CdS QDs.32,35 From the data of agarose gel electrophoresis (Figure 3a), the molecule number of HisRLuc·GB1 protein bound to one QD particle is


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estimated to be about 6. HisRLuc protein also binds to the surface of GSH-QDs, and the molecule number of HisRLuc protein bound to one QD particle is estimated to be 11-17 (Figure S2a). The molecular number of RLuc protein binding to one QD particle affects on the emission intensity of BRET in the HisRLuc·GB1 protein and HisRLuc conjugated QDs. To estimate the molecule number of HisRLuc·GB1 protein, size-exclusion HPLC was also performed. From a linear relationship between the molecular weights of standard proteins and their retention times, apparent molecular weights of GSH-QDs and HisRLuc·GB1 functionalized QDs (GB1·RLuc-QDs) are calculated to be 230 kDa and 500 kDa, respectively (Figure 3b). Since the molecular weight of HisRLuc·GB1 protein is 45.5 kDa,

Figure 3. (a) Agarose gel electrophoresis of GSH-QDs and GSH-QD+HisRLuc·GB1, monitored at NIR fluorescence at 830 nm with excitation of 650 nm. The molar ratio of GSH-QD: HisRLuc·GB1 is varied from 1:0 to 1:6. (b) Size-exclusion HPLC chromatograph for GSH-QDs and GB1·RLuc-QDs. The inset shows a plot of retention times of standard proteins. Molecular weight: 450 kDa for ferritin, 148 kDa for antibody (IgG), 66 kDa for bovine serum albumin (BSA), and 30.8 kDa for EGFP. (c) Emission spectra of HisRLuc·GB1+CTZ and GB1·RLuc-QDs+CTZ. (d) Decay of the emission intensity at 830 nm for the GB1·RLuc-QDs in the presence of CTZ. Inset shows their emission images. ACS Paragon Plus Environment

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the molecule number of the protein binding to one QD particle is estimated to be 5.9. This result is consistent with the data of agarose gel electrophoresis (Figure 3a). To confirm BRET in GB1·RLuc-QDs, we measured emission spectra of the GB1·RLucQDs in the presence of CTZ. Compared with the emission spectrum of HisRLuc·GB1+CTZ, the spectrum of GB1·RLuc-QDs+CTZ shows a dual emission at 486 nm and 830 nm, which is resulting from BRET from CTZ to QDs (Figure 3c). In the GB1·RLuc-QDs+CTZ system, the emission intensity at 485 nm decreased two times compared to the emission intensity of HisRLuc·GB1+CTZ. The efficiency of BRET from CTZ to QD can be calculated by the following equation: E = 1- (IDA/ID) × 100 %, where ID and IDA represent the emission intensity of the donor (CTZ) in the absence and presence of acceptor (QD), respectively. 33,49,50 In the GB1·RLuc-QDs+CTZ system, strong NIR emission (at ca. 830 nm) coupled by BRET was observed with a BRET efficiency of ca. 50 %. The BRET efficiency was also quantified by the BRET ratio, which is defined as the intensity ratio of donor emission to acceptor emission intensity.15,16,30 The BRET ratio in this system was comparable to the BRET ratio reported in firefly luciferase-QD conjugate system,32 where the luciferase protein is conjugated to the QDs by six histidine tags. Figure 3d shows the time course of the BRET-coupled NIR emission in phosphate buffered saline (PBS). The life time (t1/2) of the BRET-coupled NIR emission was determined to be 98 s. Binding ability of BRET-coupled NIR-QDs to antibody. To apply GB1·RLuc-QDs to molecular imaging of EGFRs, we examined their binding ability to anti-EGFR monoclonal antibody (Ab) by using agarose gel electrophoresis. Figure 4a shows the agarose gel electrophoresis of GSH-QDs in the presence of HisRLuc·GB1 and HisRLuc·GB1+Ab. The mobility of the GSH-QDs decreased by the successive addition of HisRLuc·GB1 and Ab, indicating the binding of Ab to the surface of GB1·RLuc-QDs (Figure 4a). In contrast, the addition of Ab to the mixture of HisRLuc (without GB1 domain) and GSH-QDs did not


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Figure 4. (a) Agarose gel electrophoresis of GSH-QDs and GSH-QDs+HisRLuc·GB1 (1:20), and GSH-QD+HisRLuc·GB1+Ab (1:20:20). (b) Fluorescence correlation curves for GSH-QDs, GSH-QDs+HisRLuc·GB1 (1:20), and GSH-QDs+HisRLuc·GB1+Ab (1:20:20) in PBS. Inset shows their diffusion time. significantly change the mobility of the band of HisRLuc conjugated QDs (Figure S2b). Fluorescence correlation spectroscopy (FCS)50-52 was also used for the confirmation of the binding of GB1·RLuc-QDs to Ab. FCS measures fluorescence intensity fluctuations of fluorophores at the single molecule level, and gives the diffusion times of fluorescent molecules/particles in a confocal volume. Figure 4b shows the fluorescence autocorrelation curves for GSH-QDs, the mixture of GSH-QDs+HisRLuc·GB1 (1:20 molar ratio) and the mixture of GSH-QDs+HisRLuc·GB1+Ab (1:20:20 molar ratio) in aqueous solutions. All the autocorrelation curves were fitted to the theoretical fluctuation autocorrelation curve based on a single-component diffusion model.50,51 From the autocorrelation curves, the diffusion times were determined to be 0.432±0.05 ms for GSH-QDs, 0.728±0.01 ms for the mixture of GSHQDs+HisRLuc·GB1, and 1.15±0.07 ms for the mixture of GSH-QDs+HisRLuc·GB1+Ab. The increase in the diffusion time of GSH-QDs+HisRLuc·GB1 indicates that the hydrodynamic size of GSH-QDs is increased by the conjugation of HisRLuc·GB1 protein. Further, the addition of Ab to the mixture of GSH-QDs+HisRLuc·GB1 increased their diffusion time, showing the binding of Ab to HisRLuc·GB1 conjugated QDs. The association


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constant between GB1 domain and IgG is reported as 0.3×108 M-1.45,53 From the diffusion time (1.06±0.08ms) of a standard fluorescent particle (14 nm in diameter), we estimated the hydrodynamic diameter of the GSH-QDs and their protein conjugates: 5.7 nm for GSH-QDs, 9.6 nm for GB1·RLuc-QDs, and 16 nm for Ab conjugates with GB1·RLuc-QDs. The control data of FCS using HisRLuc protein are shown in Figure S3 and S4 with a summary of the diffusion times of GSH-QDs and their conjugates with proteins. These data clearly show that the GB1 domain presented on the surface of QDs binds Ab molecules. The binding of GB1·RLuc-QDs to Ab was also confirmed by NIR fluorescence imaging of gastric cancer cells (MKN-45),54,55 which express EGFRs on their cell

Figure 5. (a) Bright field and NIR fluorescence images of gastric cancer cells incubated with GB1·RLuc-QDs in the absence and presence of anti-EGFR antibody (Ab). (b) FACS analysis of the gastric cancer cells incubated with GB1·RLuc-QDs in the absence and presence of Ab. (c) Bright field and BRET images of gastric cancer cells (MKN-45) after incubation with GB1·RLuc-QD in the presence of Ab. Background noise in the BRET image arises from non-specific binding of the QDs to the bottom of a plastic cell-culture dish. Scale bar: 100 µm. (d) BRET images of the cell pellets (MKN-45) in the absence and presence of Ab.


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membranes. The MKN-45 gastric cells were stained by GB1·RLuc-QDs in the presence of Ab (Ab+), and significant NIR fluorescence signals were detected from the cell membranes (Figure 5a). In contrast, only weak NIR fluorescence signals were detected from the cells in the absence of Ab (Ab-). Fluorescence activated cell sorting (FACS) showed that the average intensity of the NIR fluorescence from the gastric cells in the presence of Ab is three times stronger than that of the negative control cells in the absence of Ab (Figure 5b). The weak fluorescence signals (Ab-) from the cells may have resulted from non-specific binding of GB1·RLuc-QDs to the cell membrane of gastric cancer. For

the

gastric

cancer

cells

incubated with GB1·RLuc-QDs+Ab, significant emission

BRET-coupled was

observed

NIRin

the

presence of CTZ (Figure 5c and 5d). In contrast, for the cells incubated with HisRLuc-QDs+Ab, the BRETcoupled

NIR-emission

was

not

observed (Figure S5). This finding

Figure 6. Viability of MKN-45 cells in 6 h, 24 h, and 48 h after treating with 0.05-50 nM of GB1·RLuc-QDs.

indicates the binding of Ab by the GB1 domain presented on the surface of the QDs. The cytotoxicity of GB1·RLuc-QDs was checked by using MTT assay (experimental section), and significant cytotoxicity of the QDs was not observed in the concentration range up to 50 nM used in BRET and fluorescence imaging (Figure 6).


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Figure 7. BRET-coupled NIR emission (BRET) and NIR fluorescence (FL) from gastric cancer cells (0, 0.3×106, 0.7×106, 1.3×106, and 2.6×106) incubated with GB1·RLuc-QDs in the presence of anti-EGFR antibody (Ab). (a) [Ab]: 0.1 µM, [GB1·RLuc-QDs]: 5 nM. (b) [Ab]: 1 µM, [GB1·RLuc-QDs]: 50 nM. BRET-emission was measured by adding CTZ to a final concentration of 50 µM. NIR fluorescence was measured by excitation at 480 nm. Exposure time was 5 min both for BRET-coupled NIR emission and NIR fluorescence with a band path filter of 830±20 nm. Emission intensities of BRET and fluorescence are summarized in bar graphs. BRET-based NIR detection of EGFRs in living cells. To demonstrate the capability of antibody and GB1·RLuc-QD conjugates, we show highly sensitive detection of EGFR expressing gastric cancer cells (MKN-45)56 by using BRET-coupled NIR emission. We compared the detection sensitivity of the gastric cancer cells by BRET-coupled NIR emission and NIR fluorescence of QDs, where the gastric cancer cells were incubated with anti-EGFR antibody (Ab) and GB1·RLuc-QDs (Figure 7). The number of cells was varied from 0.3×106 up to 2.6×106 to determine the detection sensitivity. The detection limit of EGFR expressing gastric cancer cells by BRET-coupled NIR imaging was 0.3×106 of cells, while the detection limit by NIR fluorescence imaging was 1.3×106 of the cells. The intensity of NIR emission in


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the BRET imaging (upper panels in Figure 7a and b) was at least three times higher than that in the NIR fluorescence imaging (lower panels in Figure 7a and b). In the case of NIR fluorescence imaging, autofluorescence (background signals) increaseed with increasing the number of cells and exposure times (Figure S6). In contrast, the autofluorescence signals in the BRET imaging were not observed because of the needless of external excitation, and the background signals mainly arised from the dark current noise57 in an InGaAs devise. In the NIR imaging in BRET and FL in Figure 7, there is no significant difference in the background signals under the experimental conditions described in the caption. The higher detection sensitivity of BRET-coupled NIR imaging should be attributed to the higher efficiency of BRET emission from CTZ to QDs compared with the NIR fluorescence emission of QDs. Next, we compared EGFR expression level of cells using BRET-coupled NIR imaging, Western blotting, and FACS. We measured the EGFR expression level for five different cells: MKN-45, A431(human epidermoid carcinoma), A549 (human lung carcinoma), HeLa (human cervical carcinoma), and HEK293 (human embryonic kidney), where MKN-45, A431, A549, and HeLa cell lines were EGFR-positive, whereas HEK293 cell line was EGFR-negative.56,58 Surface plasmon resonance and immunofluorescence imaging have shown that A431 cells express highest level of EGFRs among the five cell lines.58 Figure 8a shows BRET-coupled NIR emission and NIR fluorescence imaging for the five cells (0.8×106 cells) after incubation with GB1·RLuc-QDs in the presence of anti-EGFR antibody. As a control experiment, normal human IgG59 (non-specific IgG) was used to check non-specific binding of anti-EGFR antibody conjugated GB1·RLuc-QDs to cells. The intensities of BRET-coupled NIR emission from five cells were about three times higher than those of NIR fluorescence emission (Figure 8a and Figure S7). This difference in the NIR emission between BRET and fluorescence imaging was similar with the result observed in Figure 7.


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Figure 8. (a) BRET-coupled NIR emission and NIR fluorescence (830±20nm) from different cell lines (MKN-45, A431, A549, HeLa, and HEK293 cells, 0.8x106cells/well) incubated with GB1·RLuc-QDs in the presence of anti-EGFR antibody and normal human IgG. Normal human IgG was used as a control. BRET-coupled NIR emission (BRET) was detected for 5 min with a band path filter of 830±20nm by adding CTZ to a final concentration of 50 µM. (b) Emission intensity (830±20nm) of BRET from the five cells. (c) The EGFR expression level of each cells, which was determined by Western blotting analysis. Staining was also carried out with an anti-b-actin antibody to ensure that similar amounts of samples were loaded. The bar graph shows the relative intensity of chemiluminescence (CL) for EGFR/ b-actin observed in the Western blotting.

Figure 8b summarizes the emission intensity of BRET from the five cells, showing that the EGFR expression level is A431>>A549~HeLa> MKN-45>HEK293.58 This result is almost similar with the result obtained from the Western blotting analysis (Figure 8c). Although weak BRET emissions (lower panel of Figure 8a) resulting from non-specific binding of normal human IgG to five cells were observed, EGFR expression level could be evaluated by the BRET imaging using anti-EGFR antibody and GB1·RLuc-QDs conjugates. The reliability of the BRET imaging for the evaluation of EGFR expression level was also confirmed by FACS. The FACS result shows that the EGFR expression level is A431> A549~HeLa >> MKN-45~HEK293 (Figure S8).


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Antibody-QD conjugates are most popular QD probes for molecular imaging. To prepare antibody-QD conjugates, cross-coupling reactions between QDs and antibody molecules are usually employed.40 However, most of the cross-coupling reactions result in non-specific binding between antibody molecules and QDs, leading to the aggregation of QDs.60,61 The use of adaptor proteins for antibody conjugation to QDs is an alternative method for the preparation of antibody-QD conjugates. In this work, we used HisRLuc·GB1 protein (45.5 kDa) which contains an immunoglobulin binding domain (GB1) for antibody conjugation. The HisGB1 protein (MW: 9.3 kDa) was two to three times smaller than previously reported adaptor proteins,62,63 which would lead to lower steric hindrance for the binding of antibody. Since the HisRLuc·GB1 protein directly binds to the surface of CdSe/ZnS and CdSeTe/CdS (core/shell) QDs,53,64 the conjugation of HisRLuc·GB1 proteins to the QD is very easy and rapid. The resulting HisRLuc·GB1 protein conjugated QDs can bind monoclonal IgG antibody at their surface (Figure 3a, b). The affinity of a GB1 protein to monoclonal IgG antibody is very high and their binding constant is ~108 M-1.45,53 Thus, the conjugates between IgG antibody and GB1·RLuc-QD can be used as fluorescent probes for molecular sensing as well as bioimaging (Figure 5a, 5b). In addition, the antibody conjugates with GB1·RLuc-QDs can be also used as BRET-based molecular imaging probes26,29,34,64 (Figure 5c, 5d,7,8). CONCUSIONS In this paper, we have presented NIR optical detection of EGFRs in living cells using BRET in GB1·RLuc-QDs. The present BRET-coupled NIR QDs are easily prepared only by mixing GSH-QDs and HisRLuc·GB1 protein in aqueous solutions. NIR optical detection sensitivity of EGFRs by using BRET-coupled NIR emission is at least three times higher than that by using NIR fluorescence. Although number of works on BRET-coupled QD/nanoparticles15-34 have been reported in the past decade, there are only a few papers that


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report the application of the BRET-coupled QDs as molecular imaging probes.26,29,34,64 Since the present BRET-coupled QDs (GB1·RLuc-QDs) have antibody binding domains at their surface, a variety of antibody can be conjugated to the QDs. The conjugates between antibody and BRET-coupled NIR-QDs will be used for a variety of molecular imaging in vitro and in vivo.

EXPERIMENTAL SECTION Preparation of HisRLuc·GB1 conjugated GSH-QDs (GB1·RLuc-QDs). Two hundred µL of HisRLuc·GB1 (1mg/mL, PBS) was added to 0.4 mL of an aqueous solution of GSH-QDs (1µM, 10 mM Na2CO3 solution). Then, the solution buffer was exchanged with PBS by using a gel filtration column (PD-10 columns, GE Healthcare). Characterization of recombinant proteins and QDs. The purity of recombinant proteins (HisRLuc and HisRLuc·GB1) was checked by SDS polyacrylamide gel electrophoresis. Recombinant proteins (1μg/lane) were run on a 5-20 % polyacrylamide gel (Extra PAGE one Precast gel, Nacalai Tesque) in Tris-glycine-SDS buffer, 200 V for 40 min and stained with Coomassie Brilliant Blue (CBB Stain One Super, Nacalai Tesque). A size marker (Precision Plus Protein Standard, BIO-RAD) was used to compare between recombinant protein. The expected size of HisRLuc and HisRLuc·GB1 were 40.0 kDa and 45.5 kDa. The binding of HisRLuc·GB1 to GSH-QDs and Ab to GB1·RLuc-QDs was confirmed by agarose gel electrophoresis. GSH-QDs and the mixture of GSH-QDs (1 µM) /HisRLuc·GB1 （1mg/mL）or GSH-QDs (1 µM) /HisRLuc·GB1（1 mg/mL）/Ab (1 mg/mL) were run on 1 % agarose gel in Tris-Acetate buffer (pH 8.0), 100 V for 15 min. Emissions of the QD bands were monitored at 830±25 nm. Fluorescence and bioluminescence spectra were measured with a photonic-multichannel multichannel analyser (C10027, Hamamatsu Photonics, Japan). For the fluorescence


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measurement, a 150 W-Xenon lamp was used as an excitation light source at 488 nm. For the For the bioluminescence measurement, 10 µL of CTZ (1 mg/mL) was added to the aqueous aqueous solution (1 mL) of HisRLuc·GB1 protein (20 nM). Fluorescence autocorrelation curves were measured on a compact FCS system (C941301MOD, Hamamatsu Photonics, Japan) at excitation of 473 nm using a LD pumped solidstate laser. The size of pinhole was 25 µm, and the spectral range of detection wavelengths was 500-900 nm. For the determination of the concentration of GSH-QDs, the number of QD particles in a 10 µL solution was measured by using FCS, and the QD concentration was estimated by using a 20 nM solution of Rhodamine 6G as a reference. The hydrodynamic diameters of GSH-QDs, GB1·RLuc-QDs, and their antibody complex were determined from the diffusion time (1.06±0.08 ms) of standard fluorescent beads (14 nm in diameter).51,52 The morphologies of QDs were observed by TEM using a Hitachi H-800 microscope operating at an acceleration voltage of 200 kV. The TEM sample (1 µM QDs in PBS) was prepared by dropping the sample solution onto a copper grid.

Cell viability. Human gastric cancer cells (MKN-45) were incubated with GB1·RLuc-QDs (0.05–50 nM, PBS) for 6 h, 24 h, and 48 h. MTT assay was performed according to the procedure of a MTT Cell Count Kit (Nacalai Tesque). The MTT reagent was added to each well and the cells were incubated for 2 h at 37 ℃. Then, the STOP solution was added to stop the reaction. According to the instruction of the Kit, the absorbance at 570 nm and 650 nm of solubilized MTT formazan products were measured with a Microplate Spectrophotometer (Multiskan GO, ThermoFisher). Cellular imaging. Cellular imaging was performed using a fluorescence microscope, BZX700 (KEYENCE CORPORATION, Japan). MKN-45 cells were seeded to cell culture dishes (353001, Falcon 35 mm), and incubated in RPMI-1640 medium with 10 % Fetal


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Bovine Serum (FBS) for two nights at 37 °C. Anti-EGFR monoclonal antibody was added to the cell and incubated for 3 min at room temperature. Then, the cells were washed with PBS three times. Then, the complex of QD and HisRLuc·GB1 was added and incubate for 3 min at room temperature. Then, the cells were washed with PBS three times and filled with OptiMEM (Life technologies). The cells were observed with an mPlum filter (Ex:560±40 nm /Em:590 nm LP) for QDs. BRET-emission and fluorescence measurements. The MKN-45 cells (0 to 2.6×106 cells) were incubated with antibody (0.1 µM or 1 µM) for 3 min. After the cells were washed with PBS, an aqueous solution of GB1·RLuc-QDs (5 nM or 50 nM) was added. Immediately before the detection of BRET emission, CTZ was added to the cells at a final concentration of 50 µM. BRET emission images (at 830±20 nm) were taken by using an in vivo imaging system (Bruker, MS FX PRO). Exposure time was set to 5 min. Western blotting analysis. The cells were lysed with RIPA buffer (Nacalai Tesque) for 15 min on ice and centrifuged at 10,000 × g for 10 min at 4 ℃ to remove insoluble material. Protein concentration was determined by the Quick Start Bradford 1x Dye Reagent (BIORAD). The cell lysates were separated by electrophoresis on 7.5 % Extra PAGE One Precast Gel (Nacalai Tesque) at 200 V for 45 min. The protein was transferred by Trans-Blot Turbo Transfer System (BIO-RAD). The membranes to which the protein had been transferred were cut up and down at 75 kDa of the molecular weight marker. The membranes were then incubated in 5 % skim milk for 1 hour at room temperature to prevent nonspecific binding. Then, the membranes were incubated with a primary antibody (1:100 dilution of anti EGFR A-10 antibody for the membranes more than 75 kDa, 1:200 dilution of anti β-Actin C4 antibody for the membranes of 75 kDa or less, Santa Cruz Biotechnology) in 5 % skim milk for overnight at 4 ℃. After washing with TBST, the membranes were incubated with a 1:2000 dilution of secondary antibody (Goat Anti Mouse IgG, HRP conjugate, Millipore).


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After washing with TBST, bands were visualized by treating the membranes with Luminata Forte Western HRP Substrate (Millipore) according to manufacturer’s instructions and detecting the bioluminescence with MS FX II (Bruker). ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge. Experimental details on QD and protein synthesis. Additional figures on the particle size distribution of QDs, agarose gel electrophoresis of QDs, fluorescence correlation curves of QDs, diffusion times of QDs, BRET images of gastric cancer cells, autofluorescence from gastric cancer cells, NIR emission intensity in BRET and fluorescence imaging for five different cells, and FACS analysis for the five different cells. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ORCID Takashi Jin: 0000-0003-3483-2254 Authors contribution All authors have given approval to the final version of the manuscript. T. J. and S.T. designed and conducted the experiments. T. J. and S. T. wrote the manuscript. Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENTS The authors thank Dr. Takao Sakata and Prof. Hidehiro Yasuda for their help in TEM measurements and Sayumi Yamada in cell viability analysis. ABBREVIATIONS BRET, bioluminescence resonance energy transfer; FRET, fluorescence resonance energy transfer; NIR, near infrared; QD, quantum dot; EGFR, epidermal growth factor receptor; RLuc,

Renilla

luciferase;

IgG,

immunoglobulin

G;

CTZ,

coelenterazine;

GB1,

immunoglublin binding B1 domain of protein G; HER2, human epidermal growth factor 2.


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Recombinant Protein (Luciferase-IgG Binding Domain) Conjugated

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