Bio-nano hybrid complex are materials of active interest due to its immense potential in diverse applications such as bio-mimic devices, sensors, electro-optics, biotechnology, cancer therapy and diagnostics etc [1–4]. Biomolecules, specifically optically active purple membrane (PM) have long been studied for photonic and electro-optic applications. Bacteriorhodopsin (bR) is the optically active center of the PM, which functions as a photo-induced proton pump by converting optical energy into proton gradient across the membrane [5]. bR amounts to 75% of the PM by mass, while the other 25% is due to lipids. Bacteriorhodopsin is a photosensitive seven helix transmembrane protein usually found in the halophilic bacteria, Halobacteriumsalinarium. It is a rare molecule found naturally in crystalline form with size ranging from 400 nm – 800 nm and thickness of 5-10 nm [6]. Crystalline structure provides required chemical and thermal stability to the inner bacteriorhodopsin surrounded by lipids. Due to these intrinsic properties of the PM, including high thermal stability, ability to form thin films with excellent optical and proton transport properties, the PM is widely proposed as an excellent optical material for myriad of applications [7].

Due to unique optoelectronic properties, one dimensional single walled carbon nanotubes (SWNT) are also considered as a promising material for various electro-optical, photonic devices and biosensors [8–10]. Stable donor-acceptor systems, based on optically active bio-nano hybrid complex of PM and SWNT, may play an important role in the development of next generation optical devices. Moreover, solutions of SWNTs provide opportunity to study interesting effects in colloidal solution of one dimensional particle. Stability and controlled assembly are critical challenges for colloidal systems and “bottom-up” assembly of nanoparticles in solution. This exertion describes optically induced aggregation and subsequent separation of pristine SWNTs of selective diameters, preferentially functionalized with purple membrane. Fundamental understanding of these processes should lead to a better stability, directed assembly and controlled charge transfer in donor-acceptor systems based on SWNTs.

SWNT functionalized with optically active molecules, electron-donating polymers and dyes are well- reported [11–13]; however, low solubility of SWNTs in aqueous dispersions limits its applicability, especially functionalization with bio-molecules. This limitation is overcome using bio compatible amine based surfactants. Stable aqueous dispersion of SWNTs has been formed using well established protocol of non-covalent functionalization by PL-PEG [14]. Ultrahigh Purified SWNT were purchased from NanoIntegris and were used as such without any further purification. Stable dispersions of the SWNTs were made in water using 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (C₄₁H₈₂NO₈P) powder, PL-PEG amine as a surfactant, which was used as purchased from Avanti, Polar Lipids, Inc. Stable SWNT dispersion was prepared by sonication of pristine, hydrophobic nanotubes in aqueous solutions of above mentioned amphiphilic polymer. The hydrophobic lipid chains of PL-PEG amine strongly anchors to the nanotube surface, whereas the hydrophilic amine chain provides SWNT with water solubility and biocompatibility. Prepared aqueous solutions of SWNT provides for new functionalities to be added to the nanotubes, using bio-molecules such as bacteriorhodopsin. Prepared aqueous solution of SWNTs showed excellent stability for weeks.

Optoelectronic and thin film applications demand high quantity (several milligrams) of the PM with better optical properties [15]. Hence, Halobacterium cultivation was performed in a 7 liter photo-bioreactor to give high yield of 14.4 mg/l. 79.36 micro molar (µM) from the stock protein was used for further functionalization with SWNTs. SWNTs tend to coagulate, steadily with the PM over 24 hours, in dark. However, prepared aqueous solution of PM-SWNT was irradiated horizontally through a slit, by broadband mercury white lamp (New port, oriel instruments, USA) for time periods in ratio of [1:2:3:4] starting with 1hour. Corresponding to each of the samples kept under light exposure, samples of the same concentrations were also kept in dark for similar duration. Figure 1(a) illustrates the experimental set up and conceptual model for our observation of optically induced aggregation of the functionalized SWNT dispersions. Pristine dispersions of SWNT do not show any change in concentration over the course of these experiments. Although, PM functionalized SWNTs coagulate very slowly in dark; optically induced rapid aggregation is observed for the PM functionalized SWNTs, kept under broadband illumination.

 

Fig. 1 (a) Conceptual model of light induced aggregation of PM functionalized SWNTs due to charge transfer from optically active PM. (b) Interaction and inhomogeneous surface charges leads to aggregation due to decrease in repulsive potential. (c) SEM of aggregated PM functionalized SWNTs showing random surface binding of PM with SWNTs.

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DLVO theory named after Derjaguin, Landau, Verwey and Overbeek is traditionally used to understand stability of colloidal solutions. Even with its inherent limitation, due to assumptions on size, charge dielectric properties and dimensions of interacting particles, it is widely being used to understand fundamental interactions in solutions of nanoparticles [16,17]. Bhattacharya [18] recently asked if light has the ability to aggregate particles. This is an interesting question of wider interest. Although optical effects in solutions of gold nanoparticles and functionalized SWNT have limitedly been reported, the phenomenon is not well understood [19,20]. Here, we attempt to provide theoretical frame work for our experimental observations. According to the theory, total interaction potential (V_tot) of two interacting spheres with radius R₁, R₂>>D, the inter-particle distance is given by $V_{tot}=\left(\frac{-A_H}{6D}+Z{e}^{-\kappa D}\right)\left[\frac{R_1{R}_2}{R_1+R_2}\right]$. Here, the first term is due to the attractive Van der Waals force and depends on the Hamaker constant A_H. Hamaker constant is related to the dielectric properties of the system. For ɛ_i being the dielectric constants and n_i the refractive index of SWNTs and medium (water) respectively, ν_e being the frequency of lowest electron transition:

Figure 2 illustrates absorption spectra for supernatant of optically aggregated PM functionalized SWNT dispersions. Pristine dispersions of SWNT do not show any change in concentration over the course of these experiments and very slow rate of aggregation is observed for PM functionalized SWNTs kept in dark; however, non linear, rapid aggregation is observed for the PM functionalized SWNT under a broadband illumination. Absorption spectra of SWNTs in aqueous solution show characteristic Van Hove singularities with peaks in VIS/NIR region due to semi-conductor SWNTs, and background absorption in UV and visible frequency is essentially because of plasmonic resonance of metallic SWNTs. Absorption spectra show a consistent decrease in concentration of SWNTs with illumination for different time intervals from one to four hours. To understand the dependence on frequency of illumination on peak absorption of the PM, the phenomenon was further studied using red and green band pass filters. Significantly enhanced aggregation is observed for green filter compared to red for normalized optical intensity. Rate of aggregation under red and green illumination is found to be linear, as compared to logarithmic decrease in concentration, in case of broadband illumination. Absorption spectra of the pure PM (inset Fig. 2) with molar absorption coefficient 63,000⁻¹ cm⁻¹ show broad band absorption with λ_max at 570 nm. In the range 350-470 nm, PM absorption does not exceed 20% of λ_max, whereas absorption significantly decreases beyond 600 nm into visible NIR frequency. Significantly, higher rate of aggregation for green illumination as compared to red emphasizes the dependence of phenomenon on the absorption of PM.

 

Fig. 2 (a) Absorption spectra of SWNTs functionalized with PM (bacteriorhodopsin) kept in broadband light for varying time intervals from 1 to 4 hours (top to bottom). Inset shows absorption spectra of bacteriorhodopsin with its maximum peak at 570 nm. (b) Showing optically induced rate of aggregation in PM functionalized SWNTs as compared to pristine SWNTs and PM functionalized SWNTs kept in the dark. Enhanced aggregation is seen for green light as compared to red illumination.

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Consistent, linear increase in rate of aggregation is observed with corresponding increase in the intensity of broadband illumination, as shown in Fig. 2(e). NIR absorption band of SWNTs are related to the band gap energy of the semiconducting nanotubes [25]. Hence, discernible changes in the NIR absorption of the separated supernatant with increase in duration of light exposure (Green) indicate preferential binding of the PM with selective diameters of SWNTs as shown in Fig. 2(f).

Samples were then centrifuged for 10 minutes at 5,000 rpm (Eppendorf® Mini Spin Centrifuge) to carefully separate the supernatant and aggregated floc from the solution. Raman spectrometer, labRAM, with 632 nm laser is used to further investigate the specific binding of the PM functionalized SWNT complexes. Figure 3(a) compares Radial breathing modes (RBM) of pristine SWNTs and that of aggregated floc, separated after 240 minutes of broadband visible illumination. RBM are due to a radial motion of atoms perpendicular to the axis and the resonant frequency is inversely related to the diameter of the tube by ${\omega }_{RBM}=({\alpha }_{RBM}/d)+{\alpha }_{bundle}$. Where, d is the diameters of nanotubes and α_RBM (223.5 cm⁻¹), α_bundle (12.5 cm⁻¹) are constants, respectively [25]. Histogram, as shown in Fig. 3(b) is plotted using RBM spectra, by fitting multiple Lorentzian peaks, representing individual diameters of SWNT in the pristine, control solution, and ones in the aggregated floc. As compared to pristine SWNTs, aggregated floc of SWNTs functionalized with bR shows preferential enrichment of SWNTs with the specific diameters especially of 1.45 nm, where as significant loss of intensity in RBM peaks corresponding to the larger diameter of SWNTs is observed. G and G’ band in Figs. 3(c)-(d), show specific binding and strong interaction between the bR and SWNTs to form stable functional complexes. G band in pristine SWNT shows narrow G⁺ Lorentzian peak at 1590 cm⁻¹ and broad G^-peak around 1540 cm⁻¹ due to TO (circumferential) and LO (axial) modes [26]. As reported, functionalized SWNT show changes in G^- band due to interaction between electron donors/acceptor molecules [27]. Significant increase in the characteristic G^- band is observed for functionalized SWNTs as compared to pristine SWNTs. Hence, this discernible changes in the G^- feature of the functionalized SWNT is most likely due to charge transfer and interaction between optically active bacteriorhodopsin and SWNTs. Moreover, the observed red shift of ~4-5 cm⁻¹ in G’ band in Fig. 3(d) indicates strong binding and surface interaction between the PM and SWNTs. Moreover, as expected no discernable changes are observed in the D band (Fig. 3(d) inset), confirming that this non-covalent functionalization does not affect the Sp² carbon hybridization; thus, retaining pristine electro-optical and charge transport properties of SWNTs.

 

Fig. 3 (a) RBM showing preferential binding of PM with SWNTs of specific diameters (red line) as compared to pristine SWNTs (black line). (b) Histogram as plotted using multiple peak fit in RBM showing relative enrichment in SWNTs of specific diameters. (c)G band Raman spectra showing increase in G^- band ~1540-1560 cm⁻¹ in optically aggregated SWNTs functionalized with PM (red solid line) as compared to control pristine SWNTs (black dotted line). (d)G' band shows slight red shift indicating strong binding in PM functionalized SWNTs. D band (inset)

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Understanding fundamental interactions in solutions and using controlled external forces is important for bottom-up, directed assembly of bio-nano hybrid devices. Surely, more research is needed to better understand complex interactions in SWNT functionalized with an optically active PM. However, our results confirm optically induced aggregation and consequent separation of the PM functionalized SWNT of selective diameters from solution. Enhanced aggregation is observed for optical frequency close to peak absorption frequency of bR, indicating strong interaction between the optically active PM and SWNT. The aggregated floc shows enrichment of SWNTs of specific diameters, indicating selective binding of PM with specific SWNTs. These stable bio-nano hybrid complexes based on SWNTs functionalized with optically active PM indicate promising potential for electro-optical applications. Moreover, the PM functionalized SWNT provides opportunity to better understand interactions in colloidal solutions of optically active, donor-acceptor systems. Moreover, this functionalized complex also provides stable system for fundamental studies on charge transfer and interaction between bio-nano hybrid interfaces in solution. Optically induced, selective aggregation of SWNTs functionalized with PM certainly promises potential application in optically induced separation of other functionalized nanoparticles from solution, too. In conclusion, we report optical induced aggregation and separation of stable bio nano hybrid complex of selective diameter of SWNTs, preferentially functionalized with PM.