Highly selective turn-on detection of (strept)avidin based on self-assembled near-infrared fluorescent probes

Abstract

Selective detection and visualization of specific proteins are important in clinical diagnostics and biological research. For protein sensing, small-molecule-based fluorescent turn-on probes are preferable because of their high sensitivity, simplicity and detection with high-throughput. Herein we demonstrated a small molecular fluorescent dye (SQ-Biotin) which can self-assemble into a non-fluorescent probe in aqueous solution for near infrared turn-on detection of avidin protein. This probe consisting of a hydrophobic squaraine (SQ) as a fluorophore and a specific and strong protein ligand (biotin) formed self-assembled aggregates in aqueous solution (fluorescence off), and the aggregates of the probe disassembled in response to the target protein (avidin) through the specific protein–ligand interaction (fluorescence on). The conversion of the aggregation of SQ-Biotin was confirmed by field emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM). The fluorescence intensities at 665 nm were linearly proportional to the concentration of avidin over the range of 0.76–1.46 μM. The detection limit was calculated to be about 70 nM. SQ-Biotin showed good selectivity to avidin over other proteins, enabling turn-on fluorescent detection of avidin in the near infrared region. The strategy demonstrated the great potential applicability of the self-assembled small-molecule-based fluorophores for protein sensing in clinical diagnosis.

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Introduction

Protein biomarkers overexpressed in disease-related cells or tissues are important targets for biological research and therapeutic application. Selective sensing and imaging of these biomarkers are favorable in medical diagnosis and pathogen detection.¹ Several fluorescence turn-on strategies such as supramolecular approaches² and polymer-conjugated nanoparticles³ have been reported for protein sensing. However, the fluorescent probes based on these macromolecules suffer from low selectivity, small fluorescent turn-on ratios and more importantly, limitation to sensing proteins only on the cell surface due to steric hindrance. In contrast, small-molecule fluorescent turn-on probes are more attractive for protein detection as their easy synthesis, fine-tuning properties and high-throughput protein imaging while keeping them in native cellular environments.⁴ Upon recognition of targeted proteins, their molecular environment experiences change. In response to the changes, turn-on fluorescence can be achieved through electron and charge transfer,⁵ proton transfer,⁶ and internal conversion due to molecular rotation.⁷ Based on these mechanisms, they can selectively detect not only enzyme but also non-enzymatic proteins. However, most of them show some crucial drawbacks. First, they are always internally fluorescent. Upon recognition of targeted proteins, they show small fluorescence change due to the existence of high noise signals of background, resulting in the low sensitivity of detection. Second, they are excited in the blue region but not in near infrared (NIR) region, a perfect window for biological application in vitro and in vivo, suffering from the photodamage from short-wavelength excitation and interference with short-wavelength fluorescence from biological background. Therefore, currently it is desired to focus on the design of small-molecule probes with nearly non-fluorescence and dramatic turn on fluorescence change in NIR region before and after recognition of targeted proteins.

Squaraine (SQ), an important class of dyes with sharp and intense absorption and fluorescence in the red to near infrared region, are extensively used as probes for the detection of cations,^8,9 and neutral molecules.¹⁰ But for protein sensing, SQ is still seriously limited to human/bovine serum albumin (HSA/BSA) through hydrophobic interaction.¹¹ Selective detection of other proteins using NIR SQ dyes continues to be a challenge. In the other hand, SQ dyes exhibit a high tendency to form aggregates via strong π–π conjugated interaction in aqueous solution. Accordingly, SQ dyes show nearly non-fluorescent on account of their aggregation-caused quenching (ACQ) property.^11e,12 These properties make SQ to be an ideal candidate of fluorophore for fluorescent probe because they will demonstrate large signal-to-noise response to targets. Very recently, Klymchenko and co-workers used SQ-based far-red probes to obtain a very high turn-on response through the polarity-sensitive folding of its dimmers.¹³ Although these probes have been applied for bioimaging, they are needed elaborate synthetic procedures and not used for proteins sensing. Alternatively, we herein introduce a novel strategy to generate SQ NIR fluorescent probes readily self-assembled to probes in aqueous solution for selective detection of avidin, a protein that more frequently occurred in tumor cells.¹⁴ The probe (SQ-Biotin) consists with a hydrophobic squaraine (SQ) fluorophore and a protein–specific ligand (biotin) (Scheme 1). The probes form self-assembled aggregates in aqueous solution (fluorescence off) and convert disassembly (fluorescence on) in response to the target protein (avidin). The recognition-based disassembly is driven by the strong affinity between biotin and avidin. As the aggregation equilibrium based on the π–π conjugated interaction is disrupted, SQ dyes will deaggregate to monomer and their quenched fluorescence would recover, resulting in a sensitive fluorescence turn-on in the NIR region for recognition of avidin. Our probes combine the following merits into a signal molecular assembly. First, preparation of our fluorescent probes require only simple synthetic steps to introduce a simply linker group (ethanediamine) to connect the fluorophore (SQ) and the protein ligand.² Although similar strategies based on rhodamine nanoaggregates have been used for protein sensing,² the strict selections are needed for the linker groups between dye and recognition ligand to ensure self-assembly. But in the case of SQ-based aggregates, simple link groups are enough to form aggregates due to the strong hydrophobicity of SQ. Thereby, the design and synthesis procedures for these probes are greatly simplified. Second, the self-assembled probes which are nonfluorescent in aggregate state can background-freely detect protein with large signal-to-noise ratio.² Although environment-sensitive probes with suitable ligands are other alternations for detection of protein,⁴ these environment-sensitive fluorophores are not only extremely scare but also self-fluorescent, which limit their broad application. In contrast, SQ, like most of fluorescent dyes, has common ACQ property. It provides the possibility that this method is general for many other dyes. Third, the probes exhibit their turn-on response in NIR region which is ideal window for avoiding short wavelength excitation-triggered photo-damage, strong interference from biological medium and scattering light.¹² By changing the recognition ligands, this approach based on SQ aggregates could be hopefully expanded to selective detection of many other proteins.

Scheme 1 Synthesis route of SQ-Biotin.

Experimental

General

All chemicals and reagents were used directly as obtained commercially unless otherwise noted. Water used was ultra filter deionized. Avidin, concanavalin, myoglobin, casein, lysozyme, RNase A, trypase, pepsin, thrombin, BSA, casein were purchased from SIGMA.

Instruments

NMR spectra were recorded on a Varian 300 Gemini spectrometer. Mass spectrum was obtained in ESI mode on a HP1100LC/MSD mass spectrometer. UV-Vis spectra were acquired on a Shimadzu 1750 UV-visible spectrometer. Fluorescence spectra were obtained on a RF-5301 fluorescence spectrometer (Japan).

Measurement procedures

The stock solutions of SQ-Biotin with a concentration of 5.0 × 10⁻⁴ M were prepared first by dissolving the appropriate amount of the dye in DMSO, respectively. For measurement of spectroscopic properties, 30 μL of each stock solution were diluted with PBS (0.01 M, 3 mL, pH 7.2) to obtain aqueous solution of SQ-Biotin (5.0 × 10⁻⁶ M) under vigorous stirring at room temperature. Stock solutions of the various proteins were prepared in deionized water and the concentrations were fixed at 6 × 10⁻⁴ M.

Atomic force microscopy (AFM) and field emission scanning electron microscope (FESEM)

Samples for the imaging were prepared by spin-casting the SQ-Biotin in the absence and presence of its specific protein (Avidin) at the specified concentrations. AFM (NanoscopeV) was performed in the ambient air condition in the tapping mode, a frequency near resonance. The scan rate was 1 Hz with a scan field of view of 500 nm × 500 nm to 5 μm × 5 μm. The microstructure of the samples was analyzed by field emission scanning electron microscopy (FESEM, S-4800). All samples were dried and detected at room temperature with SE detection at 10.0 kV.

Synthesis of SQ-Biotin

SQ-NH₂ (724 mg), triethylamine (30 μL) and anhydrous DMF (15 mL) were added into a round-bottom 50 mL flask fitted with a stirrer. An anhydrous DMF solution (5 mL) of Biotin-NHS¹⁵ (136 mg, 0.4 mmol) was added dropwise at room temperature under nitrogen atmosphere and stirred overnight. The solvent was removed under reduced pressure and the residue was applied to silica gel chromatograph (by using as CH₂Cl₂/MeOH eluent) to get solid SQ-Biotin (153 mg, 0.18 mmol, 45%). ¹H NMR (500 MHz, DMSO-d₆) δ (ppm) 8.87 (s, 1H), 8.17 (s, 1H), 7.89 (dd, J = 11.5, 7.8 Hz, 2H), 7.62 (m, 2H), 7.53–7.40 (m, 2H), 7.29 (m, 2H), 6.31 (d, J = 12.7 Hz, 2H), 6.14 (s, 1H), 6.00 (s, 1H), 4.44 (q, J = 6.1 Hz, 2H), 4.26 (q, J = 5.9 Hz, 2H), 4.23–4.18 (m, 1H), 4.05–3.97 (m, 1H), 3.55 (m, 2H), 3.27–3.21 (m, 2H), 2.98–2.86 (m, 1H), 2.72–2.65 (d, J = 12.5, 5.0 Hz, 1H), 2.57–2.49 (d, J = 12.5 Hz, 1H), 2.08–2.01 (t, J = 7.0 Hz, 2H), 1.63–1.31 (m, 6H), 1.32–1.24 (m, 6H). ¹³C NMR (125 MHz, DMSO-d₆) δ (ppm) 174.13, 173.65, 163.97, 163.18, 161.12, 160.31, 157.52, 156.28, 140.77, 140.73, 128.39, 128.22, 125.40, 125.06, 123.43, 123.14, 122.45, 119.89, 117.33, 113.61, 113.46, 87.16, 86.34, 61.51, 59.69, 55.86, 43.27, 42.09, 41.63, 35.61, 28.72, 28.58, 25.61, 13.03, 12.99. HRMS (ESI+) found 701.2406 (M)⁺, calcd for C₃₆H₄₁N₆O₃S₃, 701.2396.

Results and discussion

Synthesis of probe SQ-Biotin

The compound SQ and Biotin-NHS were synthesized according to the procedure reported previously.^11d,15 The methoxyl group in SQ was replaced by one of amino groups of ethanediamine in dichloromethane to obtain the compound SQ-NH₂. Subsequently, the protein recognized ligand biotin was connected to the produced SQ-NH₂ through amide linkage. The detailed synthetic route was outlined in ESI.† The compound SQ-Biotin was fully characterized by ¹H NMR, ¹³C NMR and HRMS.

Absorption and fluorescence response to strept(avidin)

The UV-Vis absorption and fluorescence spectra of probe SQ-Biotin in the absence and presence of avidin in aqueous solution (10 mM PBS buffer, pH 7.2) were initially investigated (Fig. 1). Probe SQ-Biotin displays two absorption bands at 600 (ε = 3.3 × 10⁴ M⁻¹ cm⁻¹) and 648 nm (ε = 5.4 × 10⁴ M⁻¹ cm⁻¹), which are attributed to aggregate and monomer, respectively. SQ-Biotin consists of hydrophobic squaraine segment and a recognition ligand biotin. In aqueous solution, probe SQ-Biotin is inclined to aggregate through intermolecular π–π and hydrophobic interaction. Upon addition of avidin, the absorption at 600 nm decreased, and the absorption peak at 648 nm gradually increased. The results suggested that SQ-Biotin underwent the conversion from aggregate to monomer after addition of avidin. Avidin, a water-soluble macromolecular protein, can selectively interact with avidin by strong affinity (K_a ∼ 10¹³ to 10¹⁵ M⁻¹).¹⁶ In the presence of avidin, the protein–specific ligand (biotin) enables SQ-Biotin to bind with avidin. Accordingly, SQ-Biotin was passively dissolved in aqueous solution and the initial self-assembled aggregates were transformed into monomer due to the equilibrium collapse of π–π and hydrophobic interaction. In fluorescence spectra excited at 600 nm, probe SQ-Biotin shows non-fluorescent in aqueous solution owing to the ACQ effect. Upon addition of avidin, the fluorescence intensity at 664 nm gradually increased. As 7.69 μM avidin were added, more than 9.12-fold increasing of fluorescence intensity was found. The fluorescence intensity at 664 nm went up linearly with the increase in concentration of avidin between 0.76–1.46 μM (Fig. 1c). The detection limit for avidin is calculated to about 70 nM, which is comparable to other reported avidin assays.⁸ The same spectra changes of SQ-Biotin upon addition of streptavidin were also observed (Fig. S1†).

Fig. 1 Spectroscopic analyses of avidin–specific SQ-Biotin. (a) UV-Vis absorption spectral changes of SQ-Biotin (5 μM) upon addition of avidin (0–2.64 μM). (b) Fluorescence spectral changes of SQ-Biotin (5 μM) upon addition of avidin (0–7.69 μM) (λ_ex = 600 nm). Fluorescence measurements were performed 1 min after adding avidin to the SQ-Biotin solution. (c) Plot of the relative fluorescence intensity (I/I₀) of the solution to avidin concentrations (0.76–1.46 μM), where I and I₀ stand for the fluorescence intensity at 664 nm in the absence and presence of avidin. All experiments were performed in 10 mM PBS buffer (pH 7.2).

The fluorescence changes of SQ-Biotin in organic solvent were investigated to prove the hydrophobic nature of quenched probes. In buffer solution, SQ-Biotin tends to form aggregates due to its hydrophobic nature, showing weak fluorescence. With the ratio of organic solvent (CH₃OH) to water increasing, the fluorescence intensity of SQ-Biotin increased. Organic solvents increase the solubility of SQ-Biotin to disassemble the aggregates of probe (Fig. S2†), resulting in turn-on response. These results prove that it is the hydrophobic nature of probes to induce the fluorescence quenching in aqueous system.

To further identify that the disassembly of SQ-Biotin is driven by the interaction between recognition ligand (biotin) and avidin, control experiment was performed. The fluorescence changes of the SQ without biotin segment upon addition of avidin were first investigated. Like the response of SQ to BSA, bathochromic shift and turn-on response were observed (Fig. S3†). The hydrophobic cavity of avidin can accommodate SQ. The binding interaction induces the rigidity of SQ to increase, resulting into bathochromic shift and turn-on response of its fluorescence.^11d For SQ-Biotin, only fluorescence turn-on but with no any fluorescence wavelength shift was observed after addition of avidin. Although the possible weak interaction between the hydrophobic cavity of avidin and SQ segment in SQ-Biotin cannot absolutely exclude, the strong affinity between biotin and avidin should be the main driving force to disassemble SQ-Biotin aggregates. In addition, another control experiment was performed. Avidin was first pretreated with biotin, then the treated avidin was added to the solution of SQ-Biotin in identical conditions described above. This treatment was expected to block the receptors of avidin and prevent competitive binding by SQ-Biotin. As expectation, no turn-on fluorescence was detected (Fig. 2). The pretreated avidin does not have the ability to disassemble the aggregate of SQ-Biotin. This result clearly proves that the interaction between probe and avidin comes from the affinity of the ligand segment of probe with avidin.

Fig. 2 The fluorescence spectra change of SQ-Biotin (5 μM) in the absence and present of avidin (3.85 μM), and SQ-Biotin (5 μM) treated with avidin (3.85 μM) in the presence of biotin (77 μM).

Aggregation characterization by AFM and FESEM

From the UV-Vis absorption spectra change of SQ-Biotin before and after addition of avidin, we have found that avidin successfully driven the self-assembled probes of SQ-Biotin from aggregate to monomer. The turn-on fluorescence response of SQ-Biotin to avidin is consistent with the disassembly process. To further validate the possible change, the self-assembly/disassembly processes of SQ-Biotin were studied by morphological transition. Atomic force microscopy (AMF) (Fig. 3) confirms that the interaction with avidin strongly affects the aggregate. The size of the SQ-Biotin aggregates obviously becomes smaller in the presence of avidin. The results clearly point to that the fluorescence turn-on is due to the structural changes of aggregation, resulting from its interaction with proteins. In addition, field emission scanning electron microscopy (FESEM) was used to check the change. As shown in Fig. 4, the morphological alteration of SQ-Biotin from blocks of aggregation (1.5 μm) to small particles (750 nm) was observed after addition of avidin. Based on these, we can present the possible fluorescence off/on switching mechanism. First, SQ-Biotin self-assembles to form submicrometer-sized aggregates in aqueous solution, which is nearly non-fluorescent due to its ACQ property. Then, the aggregates disassemble in the presence of avidin through the ligand moiety binding of SQ-Biotin to the ligand-binding domain of the target protein, correspondingly, the fluorescence enhancement can be detected in the disassembly process.

Fig. 3 AFM images of SQ-Biotin. (a) SQ-Biotin alone. (b) SQ-Biotin and avidin. The solution concentrations used for film preparation are 5 μM for the SQ-Biotin and 7.69 μM for avidin in PBS buffer and the scale is 2 μM.

Fig. 4 FESEM images of SQ-Biotin. (a) SQ-Biotin alone. Inset: the partial enlarged image. (b) SQ-Biotin and avidin. The solution concentrations used for film preparation are 5 μM for SQ-Biotin and 7.69 μM for avidin in PBS buffer. Scale bar: 20 μm.

Selective protein detection

We also examined the response of SQ-Biotin to other non-targeted enzymes and proteins including pepsin, trypsin, thrombin, myoglobin, RNase A, lysozyme, casein, BSA, myoglobin and concanavalin A. The concentration of the SQ-Biotin was 5 μM, and the concentrations of other proteins were 6.07 μM. As shown in Fig. 5, avidin and streptavidin induced more than 9- and 5.5-fold fluorescence enhancement of SQ-Biotin, respectively, but other proteins did not induce any obvious spectral changes. This result proves that the assembly system is suitable for selective detection of strept(avidin). It is reported that avidin occur in tumor cells much more frequently than in normal cells.¹⁴ Thereby SQ-Biotin has the potential to use for imaging of tumor cells.

Fig. 5 Selectivity test of SQ-Biotin with nine other non-targeted proteins. SQ-Biotin (5 μM) was tested with non-targeted proteins at 6.07 μM. Bars represent relative fluorescence intensity at 664 nm. I₀ indicates the fluorescence intensity of free proteins, while I₆₆₄ indicated the fluorescence intensity upon addition of relative proteins.

Fluorescent imaging in living tumor cells

Finally, we evaluated the possibility of SQ-Biotin for fluorescent imaging of biotin receptor-positive Hela cells (Fig. 5). After Hela cells were incubated with 10 μM SQ-Biotin for 30 min, strong red fluorescence could be observed. Pretreated Hela cells with 10 μM biotin for 30 min to block the receptors in cells, then incubated with 10 μM SQ-Biotin for another 30 min. This treatment was expected to block the receptors and prevent or reduce competitive binding by SQ-Biotin. As expectation, the fluorescence intensity in cells significantly decreased in the Hela cells pretreated with biotin (Fig. 6). The findings strongly suggested that cellular uptake of SQ-Biotin in receptor-positive Hela cells occurs by receptor-mediated endocytosis.¹⁶ SQ-Biotin can permeate into cells to image the biotin-receptors and has the potential application for fluorescent imaging in living cells.

Fig. 6 Confocal fluorescence of living Hela cells incubated with SQ-Biotin (10 μM) for 30 min (a) and pretreated with 10 μM biotin for 30 min (b).

Conclusions

In summary, we have developed a new SQ-based fluorescent molecule SQ-Biotin that can detect specific protein with turn-on fluorescence signals in the near infrared (NIR) regions. The SQ fluorophore was conjugated with a protein–specific ligand. The probe can be readily synthesized with suitable amphiphilicity, which induce the self-assembly of probes in aqueous solution through π–π and hydrophobic interaction. The switching mechanism is based on the self-assembly (signal off) and protein-recognition-driven disassembly (signal on) of ligand-tethered fluorophores. This probe shows remarkable absorbance change and fluorescence enhancement to its target protein. A linear relationship between the fluorescence intensity at 664 nm and avidin concentration has been found in the range of 0.76 to 1.46 μM. Its detection limit is calculated to be about 70 nM. Moreover, SQ-Biotin responds to avidin with high selectivity over other proteins, leading to a turn-on fluorescent detection of avidin. It is expected this fluorescent turn-on approach based on aggregate/disaggregate to be a general strategy to detect diverse proteins by altering the specific ligands that on the SQ fluorophore. This approach would be applied for both the native enzymes and non-enzymatic proteins with diverse functions and structures. We believe that the present progress should facilitate the development of various target-specific probes for application in clinical diagnostics and cellular imaging.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant no. 21206137 and 21476185), the Fundamental Research Funds for the Central Universities (2014YB027 and 2452013py014), Shaanxi Province Science and Technology (no. 2014K11-01-02-06) and the Scientific Research Foundation of Northwest A&F University (Z111021103 and Z111021107).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: ¹H NMR, ¹³C NMR and HRMS spectra. See DOI: 10.1039/c5ra07185b

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