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A new diketopyrrolopyrrole-based near-infrared (NIR) fluorescent biosensor for BSA detection and AIE-assisted bioimaging
Accepted Manuscript A new diketopyrrolopyrrole-based near-infrared (NIR) fluorescent biosensor for BSA detection and AIE-assisted bioimaging Yandi Hang, Lin Yang, Yi Qu, Jianli Hua PII: DOI: Reference:
S0040-4039(14)01802-4 http://dx.doi.org/10.1016/j.tetlet.2014.10.108 TETL 45337
To appear in:
Tetrahedron Letters
Received Date: Revised Date: Accepted Date:
21 August 2014 11 October 2014 20 October 2014
Please cite this article as: Hang, Y., Yang, L., Qu, Y., Hua, J., A new diketopyrrolopyrrole-based near-infrared (NIR) fluorescent biosensor for BSA detection and AIE-assisted bioimaging, Tetrahedron Letters (2014), doi: http:// dx.doi.org/10.1016/j.tetlet.2014.10.108
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Graphical Abstract A new diketopyrrolopyrrole-based nearinfrared (NIR) fluorescent biosensor for BSA detection and AIE-assisted bioimaging Yandi Hanga , Yi Qub, ∗, Lin Yanga , Jianli Huaa, ∗
Leave this area blank for abstract info.
1
Tetrahedron Letters j o ur n al h om e p a g e : w w w . e l s e v i er . c o m
A new diketopyrrolopyrrole-based near-infrared (NIR) fluorescent biosensor for BSA detection and AIEassisted bioimaging Yandi Hanga, Lin Yanga , Yi Qub, ∗, Jianli Huaa, ∗ a
Key Laboratory for Advanced Materials and Institute of Fine Chemicals, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China b
College of Chemistry and Chemical Engineering, Shanghai University of Engineering Science, 333 Longteng Road, Shanghai 201620, P. R. China
A R T IC LE IN F O
A B S TR A C T
Article history: Received Received in revised form Accepted Available online
A new near-infrared fluorophore (DPPAM) based on diketopyrrolopyrrole was developed as bioprobe and cell stain. This bioprobe is shown to be “turn-on” response for BSA with high sensitivity and NIR emission ranged from 600 to 850 nm. AIE-assisted bioimaging also exhibited the obvious NIR signals in some special region where the dye-aggregates attached.
Keywords: diketopyrrolopyrrole near-infrared fluorescent probe AIE BSA Bioimaging
The detection of serum albumin has attracted increasing attention because it plays a significant role in maintaining plasma pressure,1 balancing nutrition, binding and transporting various compounds. 2 Moreover, the content of serum albumin in blood plasma or other biological fluids closely related to the patient's health.3 Bovine serum albumin (BSA) is extensively researched as a model protein because of its structural homology with human serum albumin (HSB). 4 Therefore, it is particularly important to explore efficient methods of BSA detection and quantification. It is well known that BSA has a negative charge (the isoelectric point is about 4.7) in water medium at neutral pH owing to its hydrophobic cavities inside.5 Consequently, dyes with cationic groups such as ammonium and imidazolium can form aggregates induced by BSA through electrostatic and hydrophobic interaction. Many fluorescent bioprobes for BSA assays have been developed by utilizing the variations in their photophysical properties based on these mechanisms.6 However, traditional fluorescent dyes usually exhibit aggregation caused quenching (ACQ) behaviors when they are in aqueous media or bound to analyses in high concentration. Since the unique photophysical phenomenon, aggregation-induced-emission (AIE) is observed by Tang and colleagues,7 a wide variety of fluorescent “light-up” sensors have been developed based on AIE mechanism.8 But the emission of mainly AIE-active probes induced by analyses usually located in the UV-Vis light region, which largely hinder their application in vivo imaging. Recently, 1,4-Diketo-3,6-diphenylpyrrolo[3,4-c]pyrrole (DPP) and its derivatives have been extensively applied in polymer solar cells (PSCs),9 OLEDs, 10 field effect transistors (FET),11 fluorescent probes,12 two-photon absorption, 13 and dye sensitizing solar cell 14 applications due to their excellent red and
———
2013 Elsevier Ltd. All rights reserved.
strongly fluorescent emission and brilliant light, weather and heat stability.15 In our previous work,16 the DPP derivatives functionalized with electron-donating methoxytriphenylamine groups were AIE-active and can exhibit red to near-infrared (NIR) emission and large Stokes shift, which are advantages in bioapplications.17 Herein, we have developed a new NIRemissive DPPAM dye (Scheme 1) with ammonium groups. The DPPAM is nearly non-emissive in aqueous solution, but it will exhibit strong emission in NIR region after formation of aggregates with BSA due to the electrostatic interactions. Thence, DPPAM can be used as a fluorescent light-up bioprobe for BSA detection. Additionally, the ammonium groups induced to DPP core also ameliorate its water solubility, which expand its application in bioimaging.
Scheme 1. Synthesis of target compound DPPAM
The synthesis of target compound DPPAM is shown in Scheme 1. In the first step, 1,4-dibromobutane was attached to core DPP to form compound 2. This was followed by palladiummediated Suzuki cross-coupling reaction between compound 2 and 4-(bis(4-methoxyphenyl)-amino)phenyl boronic acid, which
∗ Corresponding author. Tel.: +86-21-64250940; fax: +86-21-64252758; e-mail: [email protected]; [email protected]
2 led to compound 3. Subsequently, treatment of compound 3 with trimethylamine (TMA) in anhydrous tetrahydrofuran (THF) gave DPPAM. All the new compounds were well characterized by 1H NMR, 13C NMR, and HRMS (Supporting Information).
nonluminescence. In the mixed solvent of DMSO/H2O (v/v = 1/99), the absorption spectrum of DPPAM showed a leveled-off tail in the visible region, which resulted from the formations of the aggregates of DPPAM (Figure S1a). But the emission was as faint as its initial DMSO solution. This phenomenon can be construed as that the DPPAM molecules can not be aggregated well due to its amphiphilic nature in aqueous mixtures. However, the fluorescence emission of its solid could be observed (Figure S1b), implying DPPAM is AIE active.
Figure 2. Particle size distribution of DPPAM in absence (a) and presence (b) of BSA (50 µM) in DMSO/PBS buffer (v/v = 1/1) mixtures studied via DLS and SEM (inserted images)
Figure 1. (a) UV-absorption of DPPAM (10 µM) in the absence and presence of BSA (100 µM) in PBS buffer (containing 137 mM sodium chloride, 2.7 mM potassium chloride and 10 mM phosphate buffer, 50% DMSO, pH = 7.4). (b) Fluorescence (FL) titration spectra of DPPAM (10 µM) in the presence of increasing BSA (from bottom to the top curve, 0, 0.03, 0.05, 0.07, 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2.0, 3.0, 5.0, 7.0, 10.0, 15.0, 30.0, 50.0 and 100.0 µΜ). (c) Plotting the FL intensity as a function of BSA concentration for determination of the limit of detection (LOD) of DPPAM. Insert (a): photographs of DPPAM solution before and after addition of BSA under natural light. All FL spectra were measured in DMSO/PBS buffer (v/v = 1/1) with excitation at λex = 530 nm.
The AIE properties of DPPAM were investigated by absorption and fluorescence (FL) spectra (c = 1 × 10-5 M, Figure S1). DPPAM in DMSO solution exhibited practically
In order to profile the complexion of DPPAM and BSA, absorption of DPPAM with BSA were firstly examined by adding 10 equivalent of BSA into DPPAM solution. As shown in Figure 1a, the absorption maxima of DPPAM was appeared at 521 nm and 532 nm in the absence and presence of BSA, respectively, which was bathochromically shifted by 11 nm. Meanwhile, an apparent color change from red to purple in ambient light can be obviously observed by naked eye as shown in insert Figure 1a. The absorption spectra of DPPAM broadened and showed a leveled-off tail, which indicated the aggregation of luminogen in the presence of BSA, as known as the Mie effect.7 This signified the complexion of DPPAM and BSA was occurred. Therefore, further fluorescence titration experiment was carried out. Figure 1b showed the variation of FL spectra of DPPAM after addition of different concentrations of BSA. As expected, fluorescent intensity of DPPAM enhanced gradually by increasing the concentration of BSA and ultimate enhancement factor was about 30-fold. Meanwhile, its emission maxima was appeared at 703 nm (NIR region), with a large Stokes shift of 171 nm and the peak tail reached 850 nm. Moreover, in the BSA concentration range of 0–3.5 µM, the plot of the FL intensity at 703 nm as a function of BSA concentration (c) was a linear line as shown in Figure 1c. The detection limit
3 for DPPAM was measured to be ≈ 180 nM (3δ per slope). It became obvious that the DPPAM could be used as a light-up bioprobe for quantitative detection of BSA. In order to further verify that the enhancement of the fluorescence intensity of DPPAM is ascribed to formation of aggregates of DPPAM with BSA, the formation of aggregates were investigated with a Scanning Electron Microscopy (SEM) for DPPAM before and after addition of BSA and their corresponding size were studied using dynamic light scattering (DLS). As can be seen, the aggregates of DPPAM with BSA were obviously observed from SEM images and its mean diameter was approximately 824 nm, which was much larger than the particle size of DPPAM in the absence of BSA (Figure 2). All of these analyzes above revealed that aggregates were indeed formed in the solution of DPPAM containing 5.0 equiv of BSA. Hence, the NIR emission of DPPAM was indeed induced by aggregation formation of the complexion of ammonium groups of DPPAM with BSA due to the electrostatic interactions (Scheme 2).
Scheme 2. Illustration of the fluorescence turn-on sensor for BSA based on the AIE feature of DPPAM
Figure 4. Confocal laser scan imaging of Hela cells with DPPAM incubated in PBS, pH 7.4, 37 oC. (a) bright field, (b)dark field and (c) merged images of Hela cells incubated with DPPAM (10 µM) for 45 min. Ex = 515 nm, Em collected: 600-700 nm.
The NIR emission and large Stokes shift of DPPAM with BSA has shown potential utility of DPPAM in intracellular imaging. To examine whether DPPAM can enter cells and image, bioimaging with DPPAM was investigated in HeLa cells with a confocal laser scanning microscopy (CLSM). The DPPAM was prepared in 1% DMSO PBS buffer (pH = 7.4) and the HeLa cells were incubated with DPPAM (10.0 µM) for 45 min at 37°C. As shown in Figure 4, bright red/NIR fluorescence can be observed in Hela cell, demonstrating that DPPAM aggregates was biocompatible and stained living cells. In addition, DPPAM distributed uniformly in cytoplasm also indicated that DPPAM can easily enter living cells which were further confirmed by the Z-Scan and 3D model images (Figure S4). Clearly, DPPAM can be used as a new NIR fluorescent stain for intracellular imaging. In summary, we have developed a new NIR emission bioprobe (DPPAM) based on DPP functionalized with ammonium groups for detection of BSA. DPPAM is AIE-active and exhibits a distinct enhancement of NIR emission around 703 nm owing to aggregation formation of the complexion of ammonium groups of DPPAM with BSA through the electrostatic interactions. The aggregation was demonstrated by UV-vis spectra, DLS and SEM analyses of DPPAM with and without BSA. The improvement of water-solubility of DPPAM by introducing ammonium groups also makes it useful in bioimaging. In this study we provide a simple method to design and synthesize NIR bioprobe based on DPP derivatives for detection of proteins and bioapplications. Acknowledgement. We thank the 973 project (2013CB733700 and 2013CB834701), the National Natural Science Foundation of China (21372082, 2116110444, 21172073). Supplementary data: Supporting Figures S1- S4, and compound characterization data (1H NMR, 13C NMR, and ESI-TOF mass). References 1. 2. 3.
4. 5. 6. Figure 3. FL intensity variation of DPPAM (10 µM) in the presence of various proteins (30 µM) in PBS buffer (containing 137 mM sodium chloride, 2.7 mM potassium chloride and 10 mM phosphate buffer, 50% DMSO, pH = 7.4, protein from left to right: blank, BSA, heparin, lysozyme, cytochrome C, RNase A, trypsin.
Next, the selectivity experiment of DPPAM towards BSA was carried out. The addition of several other common proteins including lysozyme, cytochrome C, RNase A, trypsin, did not confer any remakable FL change to DPPAM. Only the addition of heparin which also contain negative charge led to similar but slight FL enhancement as shown in Figure 3. Thus, the DPPAM displays relatively high selectivity for BSA in the presence of other proteins.
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S0040-4039(14)01802-4 http://dx.doi.org/10.1016/j.tetlet.2014.10.108 TETL 45337
To appear in:
Tetrahedron Letters
Received Date: Revised Date: Accepted Date:
21 August 2014 11 October 2014 20 October 2014
Please cite this article as: Hang, Y., Yang, L., Qu, Y., Hua, J., A new diketopyrrolopyrrole-based near-infrared (NIR) fluorescent biosensor for BSA detection and AIE-assisted bioimaging, Tetrahedron Letters (2014), doi: http:// dx.doi.org/10.1016/j.tetlet.2014.10.108
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Graphical Abstract A new diketopyrrolopyrrole-based nearinfrared (NIR) fluorescent biosensor for BSA detection and AIE-assisted bioimaging Yandi Hanga , Yi Qub, ∗, Lin Yanga , Jianli Huaa, ∗
Leave this area blank for abstract info.
1
Tetrahedron Letters j o ur n al h om e p a g e : w w w . e l s e v i er . c o m
A new diketopyrrolopyrrole-based near-infrared (NIR) fluorescent biosensor for BSA detection and AIEassisted bioimaging Yandi Hanga, Lin Yanga , Yi Qub, ∗, Jianli Huaa, ∗ a
Key Laboratory for Advanced Materials and Institute of Fine Chemicals, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China b
College of Chemistry and Chemical Engineering, Shanghai University of Engineering Science, 333 Longteng Road, Shanghai 201620, P. R. China
A R T IC LE IN F O
A B S TR A C T
Article history: Received Received in revised form Accepted Available online
A new near-infrared fluorophore (DPPAM) based on diketopyrrolopyrrole was developed as bioprobe and cell stain. This bioprobe is shown to be “turn-on” response for BSA with high sensitivity and NIR emission ranged from 600 to 850 nm. AIE-assisted bioimaging also exhibited the obvious NIR signals in some special region where the dye-aggregates attached.
Keywords: diketopyrrolopyrrole near-infrared fluorescent probe AIE BSA Bioimaging
The detection of serum albumin has attracted increasing attention because it plays a significant role in maintaining plasma pressure,1 balancing nutrition, binding and transporting various compounds. 2 Moreover, the content of serum albumin in blood plasma or other biological fluids closely related to the patient's health.3 Bovine serum albumin (BSA) is extensively researched as a model protein because of its structural homology with human serum albumin (HSB). 4 Therefore, it is particularly important to explore efficient methods of BSA detection and quantification. It is well known that BSA has a negative charge (the isoelectric point is about 4.7) in water medium at neutral pH owing to its hydrophobic cavities inside.5 Consequently, dyes with cationic groups such as ammonium and imidazolium can form aggregates induced by BSA through electrostatic and hydrophobic interaction. Many fluorescent bioprobes for BSA assays have been developed by utilizing the variations in their photophysical properties based on these mechanisms.6 However, traditional fluorescent dyes usually exhibit aggregation caused quenching (ACQ) behaviors when they are in aqueous media or bound to analyses in high concentration. Since the unique photophysical phenomenon, aggregation-induced-emission (AIE) is observed by Tang and colleagues,7 a wide variety of fluorescent “light-up” sensors have been developed based on AIE mechanism.8 But the emission of mainly AIE-active probes induced by analyses usually located in the UV-Vis light region, which largely hinder their application in vivo imaging. Recently, 1,4-Diketo-3,6-diphenylpyrrolo[3,4-c]pyrrole (DPP) and its derivatives have been extensively applied in polymer solar cells (PSCs),9 OLEDs, 10 field effect transistors (FET),11 fluorescent probes,12 two-photon absorption, 13 and dye sensitizing solar cell 14 applications due to their excellent red and
———
2013 Elsevier Ltd. All rights reserved.
strongly fluorescent emission and brilliant light, weather and heat stability.15 In our previous work,16 the DPP derivatives functionalized with electron-donating methoxytriphenylamine groups were AIE-active and can exhibit red to near-infrared (NIR) emission and large Stokes shift, which are advantages in bioapplications.17 Herein, we have developed a new NIRemissive DPPAM dye (Scheme 1) with ammonium groups. The DPPAM is nearly non-emissive in aqueous solution, but it will exhibit strong emission in NIR region after formation of aggregates with BSA due to the electrostatic interactions. Thence, DPPAM can be used as a fluorescent light-up bioprobe for BSA detection. Additionally, the ammonium groups induced to DPP core also ameliorate its water solubility, which expand its application in bioimaging.
Scheme 1. Synthesis of target compound DPPAM
The synthesis of target compound DPPAM is shown in Scheme 1. In the first step, 1,4-dibromobutane was attached to core DPP to form compound 2. This was followed by palladiummediated Suzuki cross-coupling reaction between compound 2 and 4-(bis(4-methoxyphenyl)-amino)phenyl boronic acid, which
∗ Corresponding author. Tel.: +86-21-64250940; fax: +86-21-64252758; e-mail: [email protected]; [email protected]
2 led to compound 3. Subsequently, treatment of compound 3 with trimethylamine (TMA) in anhydrous tetrahydrofuran (THF) gave DPPAM. All the new compounds were well characterized by 1H NMR, 13C NMR, and HRMS (Supporting Information).
nonluminescence. In the mixed solvent of DMSO/H2O (v/v = 1/99), the absorption spectrum of DPPAM showed a leveled-off tail in the visible region, which resulted from the formations of the aggregates of DPPAM (Figure S1a). But the emission was as faint as its initial DMSO solution. This phenomenon can be construed as that the DPPAM molecules can not be aggregated well due to its amphiphilic nature in aqueous mixtures. However, the fluorescence emission of its solid could be observed (Figure S1b), implying DPPAM is AIE active.
Figure 2. Particle size distribution of DPPAM in absence (a) and presence (b) of BSA (50 µM) in DMSO/PBS buffer (v/v = 1/1) mixtures studied via DLS and SEM (inserted images)
Figure 1. (a) UV-absorption of DPPAM (10 µM) in the absence and presence of BSA (100 µM) in PBS buffer (containing 137 mM sodium chloride, 2.7 mM potassium chloride and 10 mM phosphate buffer, 50% DMSO, pH = 7.4). (b) Fluorescence (FL) titration spectra of DPPAM (10 µM) in the presence of increasing BSA (from bottom to the top curve, 0, 0.03, 0.05, 0.07, 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2.0, 3.0, 5.0, 7.0, 10.0, 15.0, 30.0, 50.0 and 100.0 µΜ). (c) Plotting the FL intensity as a function of BSA concentration for determination of the limit of detection (LOD) of DPPAM. Insert (a): photographs of DPPAM solution before and after addition of BSA under natural light. All FL spectra were measured in DMSO/PBS buffer (v/v = 1/1) with excitation at λex = 530 nm.
The AIE properties of DPPAM were investigated by absorption and fluorescence (FL) spectra (c = 1 × 10-5 M, Figure S1). DPPAM in DMSO solution exhibited practically
In order to profile the complexion of DPPAM and BSA, absorption of DPPAM with BSA were firstly examined by adding 10 equivalent of BSA into DPPAM solution. As shown in Figure 1a, the absorption maxima of DPPAM was appeared at 521 nm and 532 nm in the absence and presence of BSA, respectively, which was bathochromically shifted by 11 nm. Meanwhile, an apparent color change from red to purple in ambient light can be obviously observed by naked eye as shown in insert Figure 1a. The absorption spectra of DPPAM broadened and showed a leveled-off tail, which indicated the aggregation of luminogen in the presence of BSA, as known as the Mie effect.7 This signified the complexion of DPPAM and BSA was occurred. Therefore, further fluorescence titration experiment was carried out. Figure 1b showed the variation of FL spectra of DPPAM after addition of different concentrations of BSA. As expected, fluorescent intensity of DPPAM enhanced gradually by increasing the concentration of BSA and ultimate enhancement factor was about 30-fold. Meanwhile, its emission maxima was appeared at 703 nm (NIR region), with a large Stokes shift of 171 nm and the peak tail reached 850 nm. Moreover, in the BSA concentration range of 0–3.5 µM, the plot of the FL intensity at 703 nm as a function of BSA concentration (c) was a linear line as shown in Figure 1c. The detection limit
3 for DPPAM was measured to be ≈ 180 nM (3δ per slope). It became obvious that the DPPAM could be used as a light-up bioprobe for quantitative detection of BSA. In order to further verify that the enhancement of the fluorescence intensity of DPPAM is ascribed to formation of aggregates of DPPAM with BSA, the formation of aggregates were investigated with a Scanning Electron Microscopy (SEM) for DPPAM before and after addition of BSA and their corresponding size were studied using dynamic light scattering (DLS). As can be seen, the aggregates of DPPAM with BSA were obviously observed from SEM images and its mean diameter was approximately 824 nm, which was much larger than the particle size of DPPAM in the absence of BSA (Figure 2). All of these analyzes above revealed that aggregates were indeed formed in the solution of DPPAM containing 5.0 equiv of BSA. Hence, the NIR emission of DPPAM was indeed induced by aggregation formation of the complexion of ammonium groups of DPPAM with BSA due to the electrostatic interactions (Scheme 2).
Scheme 2. Illustration of the fluorescence turn-on sensor for BSA based on the AIE feature of DPPAM
Figure 4. Confocal laser scan imaging of Hela cells with DPPAM incubated in PBS, pH 7.4, 37 oC. (a) bright field, (b)dark field and (c) merged images of Hela cells incubated with DPPAM (10 µM) for 45 min. Ex = 515 nm, Em collected: 600-700 nm.
The NIR emission and large Stokes shift of DPPAM with BSA has shown potential utility of DPPAM in intracellular imaging. To examine whether DPPAM can enter cells and image, bioimaging with DPPAM was investigated in HeLa cells with a confocal laser scanning microscopy (CLSM). The DPPAM was prepared in 1% DMSO PBS buffer (pH = 7.4) and the HeLa cells were incubated with DPPAM (10.0 µM) for 45 min at 37°C. As shown in Figure 4, bright red/NIR fluorescence can be observed in Hela cell, demonstrating that DPPAM aggregates was biocompatible and stained living cells. In addition, DPPAM distributed uniformly in cytoplasm also indicated that DPPAM can easily enter living cells which were further confirmed by the Z-Scan and 3D model images (Figure S4). Clearly, DPPAM can be used as a new NIR fluorescent stain for intracellular imaging. In summary, we have developed a new NIR emission bioprobe (DPPAM) based on DPP functionalized with ammonium groups for detection of BSA. DPPAM is AIE-active and exhibits a distinct enhancement of NIR emission around 703 nm owing to aggregation formation of the complexion of ammonium groups of DPPAM with BSA through the electrostatic interactions. The aggregation was demonstrated by UV-vis spectra, DLS and SEM analyses of DPPAM with and without BSA. The improvement of water-solubility of DPPAM by introducing ammonium groups also makes it useful in bioimaging. In this study we provide a simple method to design and synthesize NIR bioprobe based on DPP derivatives for detection of proteins and bioapplications. Acknowledgement. We thank the 973 project (2013CB733700 and 2013CB834701), the National Natural Science Foundation of China (21372082, 2116110444, 21172073). Supplementary data: Supporting Figures S1- S4, and compound characterization data (1H NMR, 13C NMR, and ESI-TOF mass). References 1. 2. 3.
4. 5. 6. Figure 3. FL intensity variation of DPPAM (10 µM) in the presence of various proteins (30 µM) in PBS buffer (containing 137 mM sodium chloride, 2.7 mM potassium chloride and 10 mM phosphate buffer, 50% DMSO, pH = 7.4, protein from left to right: blank, BSA, heparin, lysozyme, cytochrome C, RNase A, trypsin.
Next, the selectivity experiment of DPPAM towards BSA was carried out. The addition of several other common proteins including lysozyme, cytochrome C, RNase A, trypsin, did not confer any remakable FL change to DPPAM. Only the addition of heparin which also contain negative charge led to similar but slight FL enhancement as shown in Figure 3. Thus, the DPPAM displays relatively high selectivity for BSA in the presence of other proteins.
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