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C: Physical Processes in Nanomaterials and Nanostructures

Enhanced Optoelectronics by Oriented Multilayers of Photosystem I Proteins in Dry Hybrid Bio-Solid Devices Omri Heifler, Chanoch Carmeli, and Itai Carmeli J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.8b02645 • Publication Date (Web): 07 May 2018 Downloaded from http://pubs.acs.org on May 8, 2018

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The Journal of Physical Chemistry

Enhanced Optoelectronics by Oriented Multilayers of Photosystem I Proteins in Dry Hybrid Bio-Solid Devices Omri Heifler, Chanoch Carmeli amd Itai Carmeli* O. Heifler, C. Carmeli Department of Biochemistry and Molecular Biology Faculty of Life Sciences Tel Aviv University Tel Aviv 69978, Israel

Itai Carmeli* Department of Engineering and Institute for Nano Technology, Bar-Ilan University Ramat-Gan, 5290002 Israel. *Corresponding author: [email protected]

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ABSTRACT: The use of dry functional protein films on solid state platforms is essential in variety of optoelectronic devises where aqueous environment is not favorable. The photoactive photosynthetic protein photosystem I (PS I) is a good choice to be used as an active component in optoelectronic devices as it was shown to be photo active under dry environment.1 Although the extinction coefficient of PS I is high, the absorption cross section of oriented mono-layer of PS I proteins attached to solid surfaces is limited by the 10 nanometer thickness of the layer. This limits the efficiency and potential use of the PS I for practical applications. In this work, the absorption cross section and the photovoltage were enhanced by fabrication of serially-oriented multilayers. This was achieved by cross-linking free amine residues located on the surface of PS I monolayer, to thiols located at the oxidizing side of successive PS I cysteine mutants. The process repeated in a successive manner, to obtain the desired multilayer thickness. The films were characterize by various methods such as absorption measurements, ellipsometry, AFM and Kelvine probe spectroscopy to determine the thickness, absorption coefficient, plasmonic effects and photopotential of the films. The multilayers demonstrated an enhanced photovoltage caused by the increase of absorption cross-section and the serial arrangement of the photosynthetic proteins. The technique developed here for formation of oriented dry multilayers can be utilized in hybrid bio-solid-state electronic devices in which an enhancement in single monolayer parameters, such as absorption cross section and photovoltage, is required.


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INTRODUCTION The use of biological proteins in solid-state electronics is intriguing because proteins were perfected by hundreds of millions of years of evolution to become highly efficient catalysts. Of special interest are the nano size efficient photoactive photosynthetic proteins that can serve as an active component in optoelectronic devices and have potential use in the growing field of hybrid bioelectronics. Photosystem I (PS I) is a transmembrane multi-subunit protein-chlorophyll complex that mediates vectorial light-induced electron transfer. The electron transport chain contains chlorophyll a special pair (P700), a monomeric chlorophyll a, two phylloquinones and three [4Fe-4S] iron sulfur centers (Fx, Fa/b) (Figure 1A). Light absorbed by the pigments is transferred by exciton energy transfer to the chlorophyll a special pair where charge separation occurs within a picosecond that drives electron transport across the protein. The electron transfer generates a stable pototvotage of 1 V (Figure 2B) within 200 ns with quantum efficiency of 100% and absorbed light energy conversion efficiency of 47% (or ~23% of solar radiation).2 Integration of PS I into electronic devices require the formation of functional electronic junctions between the protein and the solid surface and structural and electrical stability of the protein. The structural stability of PS I is due to hydrophobic interactions which integrates 96 chlorophyll and 22 carotenoid pigment molecules and the trans membrane helixes of the core subunits (Figure 1).3 The light-induced electron transfer at cryogenic temperatures4 is an indication of little structural motions required during the optoelectronic function of PS I. Until now, plant PS I and bacterial reaction centers were indirectly attached to solid state surfaces5,6 or intercalated in organic cells7. Most of the works to date were directed at the fabrication of electrochemical devices in which hybrid PS I solids functioned under aqueous environment.8'9'10'11 A detailed reviews of many of the contribution that deals with the fabrication of hybrid PS I-solids were


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recently described.

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In this work we aim at fabrication of efficient electronic junctions

between PS I and solid surfaces that will function under dry environment in order to be integrated in solid state dry electro-optical devices. We have shown earlier the formation of active electronic junctions between PS I cysteine mutants and metal surfaces1, semiconductor12 and carbon nanotubes1. The active junctions were achieved by covalent binding of the cysteine mutation to plain surfaces or chemically modified surfaces and nano structures. Such junctions were demonstrated to efficiently conduct current under dry environment.1 Monolayers of PS I and of various types of proteins are known to conduct current under dry environment.13 For light induced electric conductance such as the PS I, the short path length of a single protein layer limits its potential efficiency. For example, the molar absorption coefficient of the chlorophylls in photosynthetic reaction center protein PS I is very high, A680 7x104 M-1cm-1, but it absorbs less than one percent of the incoming light. This is due to the short (less than 10 nm) light path and the given density of the chlorophylls in a monolayer of PS I. Therefore, oriented multilayers can be advantageous to a monolayer when a larger light absorption cross section and enhanced photovoltage values are desired in the fabrication of electro-optical devices. In fabricated efficient oriented multilayer, the PS I complexes need to be physically and electronically coupled and organized in a serial fashion.


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METHODS Unique cysteine mutants were induced by site-directed mutagenesis in the psaB and psaC genes from Synechocystis sp. PCC 6803 resulting in Y634C/Y236C and D31C, respectively and PS I was isolated.14 The electro-optical activity of isolation of PS I was determined as earlier described.

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Monolyers of PS I were fabricated on 200 nm gold over 10 nm titanium covered

glass slides and pre-treated with dithio-bis-maleimidoethane (DTME) to form a short linker monolayer. The surface was then incubated in ~0.5 mg/ml chlorophyll PS I solution to form the first layer (Experimental S). Alternatively, PS I cysteine mutants were self-assembled directly to gold surfaces as earlier described.14 Multilayer of the PS I were fabricated layer by layer with some variations between films. PS I monolayer was first incubated in a medium containing 2 mM m-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester (S-MBS). The S-MBS binds to the amine end groups of the PS I monolayer by the N-hydroxysulfosuccinimide ester moiety. The Maleimidobenzoyl group at its free end binds to the Cys PS I mutant. In an attempt to enhance the film conductivity the modification of the monolayer by S-MBS was preceded by binding of small conducting molecules- p-phenylenediamine (PPD). PPD binds to free carboxyl residues of PS I layers by cabodiimid chemistry, as earlier described16. These multilayers where then topped with (1,2-Methanofullerene C60)-61-carboxylic acid (CF) (Figure 1A) a molecule known as a very good electron acceptor. Multilayers were also fabricated by auto platinization, a technique used to connect successive layers by small Pt junctions.17 CPD was determined by KPFM in a ‘lift mode’ in an AFM model NTMDT, equipped with a custom-made 1300-nm wavelength feedback laser. The CPD is extracted in the conventional way by nullifying the output signal of a lock-in amplifier, which measures the electrostatic force at the first resonance frequency.18 AFM


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topography and KPFM were recorded in sequential scans at a scan rate of 1 Hz; 512 lines. The samples were illuminated by diode laser 670 nm 40 mV maximum power.

RESULTS AND DISCUSSION Initially, an oriented monolayer was fabricated using cysteine mutant Y634C/Y236C in subunit PsaB of PS I from the cyanobacteria Synechosystis sp. PCC 6803.14 The mutated amino acids are located near P700 in the external membrane loops and do not have stereo hindrance when placed on a solid surface, assuring the formation of sulfide bonds and close electronic junction (Figure 1A). The photochemical properties of the isolated unique PS I mutant Y634C/Y236C

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were

similar to that of the native complex.15 The fabrication of oriented monolayers was carried out by reaction of the cysteine in the mutant PS I with gold surface to form an Au-sulfide bond as previously described.14 Alternatively, a monolayer of dithio-bis-maleimidoethane (DTME) was fabricated on the gold surface to which a PS I layer was bound. A formation of thioether bond between the cysteines in PS I and the maleimide moiety of DTME resulted in the fabrication of oriented monolayer of PS I (Figure 1A). Excess protein was washed and the monolayer was dried under nitrogen. AFM images clearly show a dense monolayer of 15-21 nm particles (Figure 1B) as expected from the size of PS I as obtained by crystallography. An analysis of the AFM surface scene was done by using WSxM program of Nanotec Electronics. Particle size of 15 nm, 21 nm and 50 nm were assigned to PS I monomer, trimer and gold, respectively. The results indicated that peak to peak distances were shortened and the number of peaks increased on formation of PS I monolayer on gold surface. A density of 3.1 x 1011 / cm2 PS I units was calculated indicating a substantial coverage of the gold surface by the proteins (Table S1).


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Figure 1. Multilayer fabrication and structure. A) Simulation images of the structure of selfassembled stacked PS I molecules, the electron transport chain in space fill model (cyano), the polypeptides (gray), the chlorophylls rods (green) the cysteine mutations (yellow). The first PS I layer is attached to a monolayer of DTMA molecule on a gold surface. Amine residues on top of PS I monolayer are cross-linked to the cysteine thiols on the bottom of a successive PS I molecules by the S-MBS and PPD is linked to carboxyl residues by S-MBS. The PS I at the top are layered with caboxyfullerens. B) AFM topography images of uncoated gold surface (a), PS I monolayer (b) and three PS I layers (c) are respectively, shown. The phase of uncoated gold (d), PS I monolayer (e) and three PS I layers (f) respectively. The bar represents a scale of 250 nm. Oriented multilayer were self-assembled layer by layer over PS I monolayer with the aid of a cross-linker molecules. The PS I in the monolayer were reacted with m-MaleimidobenzoylN-hydroxysulfosuccinimide ester (S-MBS). The succinimide moiety forms amide bond to the free amines of the amino acid residues19 on top of the monolayer and the maleimide moiety forms a thioether bond with the thiol of the mutated cysteines in successive PS I layers (Figure 2A and Figure S1). In an attempt to enhance the film conductivity, the carboxyl residues exposed at the top surface of the PS I monolayer were cross-linked to p-phenylendiamine using carbodiimide chemistry (see experimental section) prior to the cross-linking with S-MBS. The multilayer formed was then coated by caboxyfullerens which is known as a very good electron acceptor (Figure 1A).20 The surface of the multilayer consists of densely packed particles of 1521 nm as seen in the AFM scan images (Figure 1B). The size of the particles is compatible with the known size of PS I. The clear difference in phase between the gold and the monolayer and multilayer indicates that the observed surface of the multilayers consists of PS I proteins as the


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phase in the AFM scan changes with the stiffness of the surface. The stiffness of gold (dark blue) is higher than that of the PSI coated gold (light blue).

A

B

C

Figure 2. Fabrication of PS I layers on gold surface and their energy scheme. A) Simulation images of the detail structure of the cross linking by S-MBA between the amine residues at


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reducing end of PS I monolayer to the cysteine thiols of a successive PS I layer. B) Energy scheme describing the solid stat and normalize hydrogen electrode (NHE) oxidation reduction levels of Au, P700, P700* and Fa/b, designating gold surface, PS I chlorophyll a special pair in ground and exited stats and the iron-sulfur clusters, respectively. C) Image simulation of photovoltage measurements by KPFM tip over two-oriented PS I layers.

Au 2L 3L 4L 5L

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0.02

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Figure 3. The absorption spectra of PS I layers. The absorption spectra of PS I layer selfassembled on gold-coated glass slides were obtained by total absorption spectrophotometric measurement. The absorption spectra of gold and of 2 up to 5 layers of PS I were recorded (A). The absorption around 683 nm PSI maxima (B). The convolution of the data by subtraction of


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the absorption of the gold gave typical absorption spectra of PS I (C). The absorption at 683 nm linearly increased with the number of layers (D). The absorption spectra of PS I layers was obtained by total absorption spectrophotometric measurement of the coated gold slides (Figure 3A). Convolution of the absorption data gave typical spectrum of PS I with absorption maxima at 442 nm and 678 nm (Figure 3B). The absorption maximum of the plasmon of gold islands at 517 nm increased as function of layer number (a). The absorption linearly increased as function of the layer number giving an average of ~0.009 A678 per layer (Figure 3C). The absorption maxima of the gold localized surface plasmon resonance at ~517 nm can also be observed (Figure 3).

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)

Figure 4. Evaluation of PS I multilayers formation by plasmon shift. PS I layers were selfassembly on Au 2.5 nm gold island on glass. (A) The effect of PS I layers on the absorption spectra gold plasmons. (B) The shift in the absorption maxima of gold plasmons as a result of the adsorption of PS I layers. The plasmon maxima shifted as a function of the layer number. A more precise evaluation of the formation of the multilayers and its effect on surface plasmons was obtained by


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self-assembly of PS I on small gold islands with defined plasmon spectra as described in reference [13]. The plasmons formed on the 2.5 nm gold island coated glass demonstrate an increase in plasmon absorption accompanied by a shift in the absorption maxima as a result of PS I adsorption (Figure 4A).21 The wavelength, intensity, and shape of the plasmon band are sensitive to changes in the dielectric properties of the surrounding, induced by binding of PS I to the metal as well as on the distance from the metal surface. As a result, there was decay of the amplitude in the shift plasmon wavelength as function of layer number (Figure 4B). This is due to the decay of the evanescent plasmon field, extending from the metal structure into the surrounding medium.22 Indeed, the absorption maxima of the plasmon shifted according to the predicted theory (Equation S1)22. The plasmon enhancement and energy shift is supports the effective formation of the multilayers. A support for the increase film thickness of the PS I layers on gold surfaces was obtained from ellipsomatry measurements. An increase in the thickness of the layer was recorded with values of 9.62 ± 1.49 nm, 28.25 ± 0.65 nm and 41.21 ± 0.02 nm for single, two and three layers, respectively. These results can be taken as qualitative indication for increase in thickness of the film with addition of PS I layers. In order to obtain quantitative a more rigorous experiments are required.23 The optoelectronic properties of dry PS I layers were evaluated by measurements of the contact potential difference (CPD) using Kelvin probe force microscopy (KPFM). The CPD and the photovoltage were determined by a KPFM system that uses a 1300 nm wavelength feedback laser not absorbed by PS I.18 The photovoltage was determined by subtracting the CPD obtain in the dark from the CPD measured under continues illumination at wavelength of 680 nm provided by a 40 mW diode laser.


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Figure 5. Photovltage measurements of PS I multilayers. KPFM scans of photovoltage changes induced by turning light on (↑) and off (↓). (A) Gold surface, (B) Four layers under nitrogen atmosphere with PPD in between the layers and CF on top of the PS I multilayer.

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Number of layers

Figure 6. Photovoltage of oriented PS I layer. KPFM measurements of increasing number of layers. The measurements of the PS I layers was conducted under air atmosphere as described under Figure 4. Photovoltage measurements of PS I layers fabricated on gold surface modified with DTME are given in Figure 5. Illumination of PS I monolayer caused an increase of 32 mV in the


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CPD (Table 1) due to a light-induced charge separation that drives electron transfer across the reaction center, resulting in the appearance of a negative charge at the reducing end of the protein away from the gold surface. Multilayers of PS I show an enhancement in photovoltage. The multilayers were fabricated by crosslinking S-MBS with p-phenilenediamine bound at the reducing end of each PS I layer and topped with caboxyfullerenes (Figure 1). A KPFM scan of the multilayer indicated that the rise in the photovoltage reached steady state level that followed by a revisable decay on turning off the illumination (Figure 5B). The photovoltage increases as function of the number of layers (Table 1 and Figure 6). The KPFM scans indicates that the polarity of the photovoltage is in the expected direction of successive binding amine groups exposed at the reducing end (top) of one layer to the cysteine at the oxidative end (bottom) of a successive PS I layer. Table 1. Photovoltage of Oriented PS I Multilayers a Sample CPD Dark CPD light Photovoltage (mV) (mV) (mV) 1. Au 16 ± 0.20 36 ± 0.30 20 ± 0.22 2. Αu, PSIWT 542 ± 62.3 548 ± 63.4 6 ± 0.10 3.Αu, PSI, CF 138 ± 1.62 170 ± 2.00 32 ± 0.31 4. Αu, PSI, PPD, PSI, CF 237 ± 26.2 300 ± 29.1 63 ± 0.59 5. Au, PSI, 2(PPD, PSI), CF 250 ± 2.15 340 ± 3.12 90 ± 0.89 6. Au, PSI, 3(PPD, PSI), CF 253 ± 2.21 369 ± 3.56 166 ± 1.73 7. Au, PSI, 3(PPD, PSI), CF (N2) 261 ± 2.31 691 ± 4.21 430 ± 4.02 8. Αu, PSID31C, PPD, PSID31C, CF 303 ± 3.51 289 ± 2.62 -14 ± 0.18 ________________________________________________________________________ a The CPD of plain gold and gold coated with a single PS I Y634C/Y236C mutant monolayer, PS I wild type (WT), by PS I multilayers and PS I multilayers doped with the conductive molecules PPD and CF. The values are given in the dark (CPD dark) and under illumination (CPD light). The third column on the right (photovoltage) is the difference (CPD light) – (CPD dark). The left column indicate the number of molecular layers in the film and it's composition. The CPD was measured by KPFM. Each value is an average of 4 samples of 512 × 512 line scans of untreated gold and multilayers of PS I monolayer measured in the dark or in the light. The measurements were done under air atmosphere and illumination was provided by a laser at 680nm, 40 mW. When indicated (N2) atmosphere was used.


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The photovoltage of four layers reached 166 mV, and 430 mV under room and nitrogen atmosphere (samples 6 and 7, respectively Table 1). A likely explanation to this difference is that under nitrogen the electrons in the electron transfer chain do not oxidize by atmospheric oxygen. The photovoltage of gold and gold surface treated with wild type PS I (native PS I that had no cysteine mutation) was smaller than that of PS I monolayer (Table 1, samples 2,3). The photovoltage almost linearly increases as function of layer number. The increase in photovotage is attributed to the increase in the absorption cross section and the serial arrangement of PS I proteins in the layers. The binding of p-phenylenediamine between the layers and the deposition of carboxyfullerenes on top of the layers gave only slightly higher but more consistent values of photovoltage when compared to multilayers with no doping (data not shown). The bound pphenylenediamine could facilitate better electronic conduction between the PS I layers as it resembles the structure of tetramethylphenylenediamine (TMPD) which is an excellent mediator of electrons. The fabrication of multilayers containing PPD might be of used in devices which require enhanced conductivity. Carboxyfullernes having work function of ~4.4 V serve as good electron acceptors at the reducing end of the PS I layer which stabilize the photovoltage at steady state illumination. Carboxyfullerns were previously shown by us to serve as electron acceptors of PS I. 20 A correlation between photovoltage sign and direction of electron transfer was demonstrated by fabrication of multilayer composed of mutant D31C. In this mutant, the cysteine is located at the reducing end of PS I. Binding D31C to the surface therefore should give opposite electron transfer polarity compared to the Y634C/Y236C in which the cysteine mutation is at the oxidizing end. The negative photovoltage obtained with multilayers composed of D31C mutant (sample 8 Table1) is an indication that the photovoltage generated is due to


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polar electron transfer within the PS I films. This results are supported by previous measurements in which we have shown the photovoltage sign is changed when PS I monolayer on metal are oriented in opposite polarity.20 In electrochemical devices increase of the mass of partially oriented PS I layer results in increase photocurrent due to water soluble mobile electron transfer mediators. The electron transport mediator FeCN accepts the electrons from the reducing end of any PS I shuttling it to the electrode.24 However, in dry PS I layers the lack of shuttling electron mediator oblige successive orientation of PS I with the same polarity to generate increase in photovoltage. This is clearly the case in the current study which is supported by previous measurements of large photovoltage in serially oriented multilayers of PS I crystals.25 We suggest that the superposition of all the individual dipoles generated by light in the serially oriented PS I molecules induce a larger net dipole. As the layers are assembled so that the reducing ends of all the PSI units are oriented in the same direction the photoinduced dipoles will increase the substrate work function and the measured photovoltage. Quantitative description of this mechanism was already formulated by us to explain the large photovoltage generated by PS I crystals. 25

CONCLUSION Previously demonstrated cases where PS I was used in hybrid protein-solid devices were performed in aqueous environment. Generally, only partial orientation of monolayers and pact layers were obtained. Only in some of the occurrences, oriented covalent binding of a monolayer was achieved. Here, we developed a method for fabrication of active PS I material which can function in solid state device under dry environment which is required in the fabrication of variety of optoelectronic devices. It was shown that the use of cross-linkers in the self-assembly


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of PS I multilayers enable the formation of dense oriented multilayers of PS I. The method enabled stacking of the protein at a desired thickness over solid surfaces with defined orientation and polarity. Multilayers were fabricated by cross-linking of free amino acid residues exposed on the surface of PS I monolayer to cysteine mutants at the oxidizing end of PS I in successive protein layers. The thickness and structure of the multilayers was evaluated and corroborated by various methods including absorption spectroscopy, plasmon enhancement and plasmon shift, AFM topography and ellipsomatry. All the measurements indicated a correlation between film thickness polarity of the PS I subunits and photovoltage generation. The absorption cross section of the serially oriented multilayers linearly increased with the layer number providing an enhanced photovoltage. This photovoltage generation proved to be more efficient under N2 conditions where the electrons in the electron transfer chain do not oxidize by atmospheric oxygen. In addition, doping of the films with conductive molecules and toping with acceptor molecules helped to mediate interlayer electron transfer and stabilize photovoltage generation. The dry multilayers can be utilized in hybrid bio-solid-state electronic devices in which an increase in photovoltage, resulting from the larger absorption cross-section and the serialarrangement of PS I, is required. The technology developed in this work for the fabrication of oriented multilayers of proteins over solid surface can be utilized for various proteins and applied in the formation of hybrid bio solid devices.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Experimental methods, the chemistry of cross-linking, statistics of AFM measurements of PSI layers, theory of localized surface plasmon resonance.


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TABLE OF CONTENT FIGURE


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Enhanced Optoelectronics by Oriented Multilayers ... - ACS Publications

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