RUBBER-LIKE MATERIAL FOR THE IMMOBILIZATION OF PROTEINS AND ITS USE IN LIGHTING, DIAGNOSIS AND BIOCATALYSIS
20180171032 · 2018-06-21
Inventors
- Rubén Dario COSTA RIQUELME (Fürth, DE)
- Uwe SONNEWALD (Möhrendorf, DE)
- Pedro BRANA COTO (Erlangen, DE)
- Michael Dominik WEBER (Erlangen, DE)
- Marlene PRÖSCHEL (Nürnberg, DE)
Cpc classification
C12N9/1205
CHEMISTRY; METALLURGY
C07K17/04
CHEMISTRY; METALLURGY
C12N9/92
CHEMISTRY; METALLURGY
C12N9/2431
CHEMISTRY; METALLURGY
C12N9/96
CHEMISTRY; METALLURGY
C12Q1/008
CHEMISTRY; METALLURGY
B82Y5/00
PERFORMING OPERATIONS; TRANSPORTING
C08J2489/00
CHEMISTRY; METALLURGY
International classification
C07K17/04
CHEMISTRY; METALLURGY
C09K11/02
CHEMISTRY; METALLURGY
C12N9/92
CHEMISTRY; METALLURGY
C12N9/96
CHEMISTRY; METALLURGY
C12N9/12
CHEMISTRY; METALLURGY
Abstract
The present invention relates to a process of preparing a rubber-like material containing a protein immobilized therein, as well as a corresponding rubber-like material, the process comprising the steps of (a) mixing a protein, a branched polymer such as trimethylolpropane ethoxylate and a linear polymer such as poly(ethylene oxide) in an aqueous solution to form a gel, and (b) drying the gel to obtain a rubber-like material containing the protein immobilized therein, wherein the branched polymer comprises at least three polymeric branches bound to a central branching unit. The rubber-like material allows the immobilization and stabilization of a wide range of different proteins, including luminescent proteins as well as enzymes, and can particularly advantageously be used as down-converting material for light-emitting diodes (LEDs), for diagnostic applications, and in bioreactors.
Claims
1. A process of preparing a rubber-like material containing a protein immobilized therein, the process comprising the following steps: (a) mixing a protein, a branched polymer and a linear polymer in an aqueous solution to form a gel; and (b) drying the gel to obtain a rubber-like material containing the protein immobilized therein; wherein the branched polymer comprises at least three polymeric branches bound to a central branching unit.
2. A process of preparing a gel, the process comprising: (a) mixing a protein, a branched polymer and a linear polymer in an aqueous solution to form a gel; wherein the branched polymer comprises at least three polymeric branches bound to a central branching unit.
3. The process of claim 1 or 2, wherein said central branching unit comprised in the branched polymer is a C.sub.1-20 hydrocarbon moiety which is substituted with 3 to 8 substituent groups, wherein said substituent groups are each independently selected from hydroxy, carboxy and amino, optionally wherein one or more carbon atoms comprised in said C.sub.1-20 hydrocarbon moiety are each independently replaced by an oxygen atom, a nitrogen atom or a sulfur atom, and further wherein each of the at least three polymeric branches is bound to one of the substituent groups of the C.sub.1-20 hydrocarbon moiety.
4. The process of any one of claims 1 to 3, wherein said central branching unit comprised in the branched polymer is selected from a trimethylolpropane moiety, a trimethylolethane moiety, a trimethylolmethane moiety, a glycerol moiety, a pentaerythritol moiety, a pentaerythrithiol moiety, a diglycerol moiety, a triglycerol moiety, a dipentaerythritol moiety, a tetraglycerol moiety, a pentaglycerol moiety, a tripentaerythritol moiety, a hexaglycerol moiety, a trimethanolamine moiety, a triethanolamine moiety, a triisopropanolamine moiety, a propane-1,2,3-tricarboxylic acid moiety, a citric acid moiety, an isocitric acid moiety, a trimesic acid moiety, a 1,1,1-tris(aminomethyl)propane moiety, a 1,1,1-tris(aminomethyl)ethane moiety, a tris(aminomethyl)methane moiety, a propane-1,2,3-triamine moiety, a tris(2-aminoethyl)amine moiety, and a tris(carboxymethyl)ethylenediamine moiety.
5. The process of any one of claims 1 to 4, wherein said at least three polymeric branches comprised in the branched polymer are each independently a poly(alkylene oxide) having a terminal OH, OR, OCOR, COOR, or CO-(R)R group, wherein each R is independently C.sub.1-5 alkyl or C.sub.2-5 alkenyl.
6. The process of any one of claims 1 to 5, wherein the branched polymer has 3 polymeric branches bound to the central branching unit, wherein said central branching unit comprised in the branched polymer is a trimethylolpropane moiety, wherein said polymeric branches comprised in the branched polymer are each independently a poly(alkylene oxide) having a terminal OH, OR or OCOR group wherein each R is independently C.sub.1-5 alkyl or C.sub.2-5 alkenyl, and further wherein the linear polymer is a poly(alkylene oxide) having a terminal group at each of its two ends which is selected independently from OH, OR and OCOR wherein each R is independently C.sub.1-5 alkyl.
7. The process of any one of claims 1 to 6, wherein the branched polymer is a trimethylolpropane ethoxylate.
8. The process of any one of claims 1 to 7, wherein the linear polymer is a poly(ethylene oxide) having a terminal OH group at each of its two ends.
9. The process of any one of claims 1 to 8, wherein the branched polymer is a trimethylolpropane ethoxylate, and wherein the linear polymer is a poly(ethylene oxide) having a terminal OH group at each of its two ends.
10. The process of any one of claims 1 to 9, wherein the protein is a luminescent protein or an enzyme.
11. The process of any one of claims 1 to 10, wherein the process does not comprise any step of covalently crosslinking the polymers that are mixed in step (a).
12. A rubber-like material containing a protein immobilized therein, which is obtainable by the process of claim 1 or any one of its dependent claims 3 to 11.
13. The rubber-like material of claim 12, wherein said rubber-like material is obtainable by the process of claim 9.
14. A rubber-like material containing a protein immobilized therein, wherein the rubber-like material comprises a branched polymer and a linear polymer, and wherein the branched polymer comprises at least three polymeric branches bound to a central branching unit.
15. The rubber-like material of claim 14, wherein the branched polymer is a trimethylolpropane ethoxylate, and wherein the linear polymer is a poly(ethylene oxide) having a terminal OH group at each of its two ends.
16. A gel which is obtainable by the process of claim 2 or any one of its dependent claims 3 to 11.
17. A gel comprising a protein, a branched polymer and a linear polymer, wherein the branched polymer comprises at least three polymeric branches bound to a central branching unit.
18. Use of the rubber-like material of any one of claims 12 to 15 as a down-converting material for a hybrid light-emitting diode, wherein the protein immobilized in the rubber-like material is a luminescent protein.
19. A hybrid light-emitting diode comprising a light-emitting diode and a coating, wherein the coating contains one or more layers of a rubber-like material as defined in any one of claims 12 to 15.
20. A diagnostic device or kit comprising the rubber-like material of any one of claims 12 to 15.
Description
[0295] The invention is also described by the following illustrative figures. The appended figures show:
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[0336] The invention will now be described by reference to the following examples which are merely illustrative and are not to be construed as a limitation of the scope of the present invention.
EXAMPLES
Example 1
Preparation of Rubber-Like Materials Containing Proteins Immobilized Therein
Preparation of Proteins and Enzymes (Recombinant Protein Expression and Purification)
[0337] The preparation and characterization of several different luminescent proteins and enzymes was performed as shown in
[0338] In this example, various different fusion proteins (containing either a fluorescent protein or an enzyme, which is fused to a human adaptor domain such as SH2, SH3 or PABC) were used because they were readily available. In the case of luminescent or fluorescent proteins, the use of such fusion proteins is advantageous due to the increased molecular weight and an increase in stability. However, the corresponding proteins (not fused to any adaptor domain) can also be used and will give analogous results.
Preparation of Rubber-Like Materials Containing Proteins Immobilized Therein
[0339] Before the formation of the rubber-like protein-based materials, a protein-based gel is formed. As a first step, the above-mentioned solutions with the different proteins are mixed with branched and linear poly(ethylene oxide) compoundsi.e., trimethylolpropane ethoxylate (TMPE) with M.sub.n of 450 mol. wt. and linear poly(ethylene oxide) (I-PEO) with M.sub.n of 510.sup.6 mol. wt., with a mass ratio of 4:1, respectively. The terminal hydroxyl groups provide a high compatibility with the protein solution, retaining enough water molecules within network. The gel network is mainly provided by the TMPE, while the I-PEO acts as a gelation agent (Prodanov L et al., Biomaterials 2010, 31, 7758). The mass ratio is optimized for the formation of a gel-like material with only the addition of an appropriate amount of water. In the studied range of protein concentrations, the formation of the gel and the final rubber material are independent of the protein amount. The optimized mixture of protein:TMPE:I-PEO in an approximate mass ratio of 1:36:3 is best described as an initial suspension that upon strong stirring over night becomes a gel, as also shown in
[0340] As a second step, the gel is deposited via doctor-blading onto any kind of substrate like, e.g., quartz (see
Detailed Procedure for Preparing Rubber-Like Materials Containing Fluorescent Proteins Immobilized Therein
[0341] The preparation of the above-discussed rubber-like materials containing a fluorescent protein immobilized therein (see
[0342] 1.) Cloning of Recombinant Gene Constructs
[0343] To combine the different protein domains and to create the pQE-9 expression constructs the overlap-PCR method was performed. Using gene specific oligonucleotides eGFP was fused to the SH2-domain (eGFP), mCherry was fused to the SH3-domain (mCherry) and mTagBFP was fused to the PABC-domain (mTagBFP). Fluorescent proteins and protein interaction domains were separated by glycine-serine linker sequences allowing proper folding of both protein domains. The extension of the proteins results in larger and more stable fusion proteins. After PCR and gel extraction the DNA fragments were ligated into the pQE-9 E. coli expression vector that contains an N-terminal 6xHis affinity tag, using T4 DNA ligase. The right orientation of the constructs and the N-terminal in frame fusion with the 6xHis tag were guaranteed using specific restriction enzymes (see
[0344] 2.) Preparation of Fluorescent Proteins E. coli strain M15 [pREP4] harboring the appropriate plasmids (pQE-9 expression constructs all containing a N-terminal 6xHis-tag coming from the pQE-9 expression vector, Qiagen) were grown at 28 C. in Lysogeny Broth (LB) medium (Bertani G, J Bacteriol. 1951, 62(3), 293-300) containing ampicillin (200 g/ml) and kanamycin (25 g/ml) antibiotics to an optical density of approximately 0.5 at 600 nm. Recombinant protein expression was induced with 1 mM isopropyl -D-1-thiogalactopyranoside at 28 C. After 4 h of induction at 28 C., cells were harvested and frozen at 20 C. Frozen bacteria cells were thawed and lysed by lysozyme treatment and sonication. Recombinant proteins were affinity purified by Ni-NTA affinity chromatography under native conditions, according to instructions of the manufacturer (QIAGEN). The concentration of the resulting purified proteins were determined by measuring the absorption at 280 nm using a NanoDrop Spectrophotometer ND-1000 (Peqlab). The purified proteins are dissolved/stored in elution buffer (50 mM NaH.sub.2PO.sub.4, pH 8.0; 300 mM NaCl; 250 mM imidazole) until further use.
[0345] 3.) Preparation and Characterization of the Protein-Based Gels and Rubber-Like Materials
[0346] The protein-based gels are prepared as follows. As a first step, the buffer solutions with the different proteins are mixed with a branched and linear poly(ethylene oxide) compoundsi.e., trimethylolpropane ethoxylate (TMPE) with M.sub.n of 450 mol. wt. and linear poly(ethylene oxide) (I-PEO) with M.sub.n of 510.sup.6 mol. wt. Both materials were purchased from Sigma Aldrich and used as received. The mass ratio is optimized for the formation of a gel-like material that allows the further film forming by using spin-coating or doctor-blading deposition techniques. In the studied range of the protein concentrations, the formation of the gels and the final rubber-like materials are independent of the protein amount. The optimized mixture of protein:TMPE:I-PEO in a mass ratio of 1:36:3 is best described as an initial suspension that upon strong stirring (1500 rpm) under ambient conditions over night becomes a gel (see
[0347] To prepare the rubber-like material, the gels are deposited via doctor-blading onto any kind of substrate like, for example, glass slides. The doctor-blading was performed using a rectangular stamp of a thickness of 50 m that was placed onto the support. They can also be applied onto 3D substrates by introducing them into the gels. Subsequently, the films were introduced into a vacuum station under 1-10 mbar for less than 1 h. The final materials are best described as rubber-like material, which are easily pilled off from the substrate with tweezers and can be easily transferred to another substrate. The thickness of the rubber-material can be controlled either by the thickness of the stamp or by the subsequent deposition of one layer on top of another with an excellent adhesion. The thickness and roughness were measured using a profilometer DektakxT from Bruker.
Example 2
Preparation of Rubber-Like Materials Using Different Mass Ratios of Branched Polymer and Linear Polymer and Different Amounts of Aqueous Buffer Solution
[0348] The preparation of the gel and the rubber-like material according to the present invention is demonstrated using different mass ratios of the branched polymer (in this case, trimethylolpropane ethoxylate (TMPE) with an M.sub.n of 450 Da) and the linear polymer (in this case, poly(ethylene oxide) (PEO) with an M.sub.n of 5000 kDa), as shown in Tables 1 and 2 below, in the absence of a protein to be immobilized. The rubber formation is performed as described in Example 1.
[0349] In particular, the two polymers are mixed at different mass ratios as shown in Tables 1 and 2 below. Although TMPE is a low viscous liquid, the PEO does not dissolve even at high stirring conditions. To facilitate this process, several amounts of buffer solution (as otherwise used for the proteins) were added.
TABLE-US-00001 TABLE 1 TMPE PEO Buffer Rubber (mg) (mg) (L) Gel formation formation 60 5 50 Highly viscous, not processable yes 60 5 100 Highly viscous, but processable yes 60 5 150 Good viscosity to make films yes 60 5 200 Good viscosity to make films yes 60 5 300 Low viscosity to make films yes 60 5 400 Low viscosity to make films yes 60 5 500 Low viscosity to make films yes
TABLE-US-00002 TABLE 2 TMPE PEO Buffer Gel Rubber (mg) (mg) (L) formation formation 60 1 150 Low viscosity to make films 60 5 150 Good viscosity to yes make films 60 10 150 Good viscosity to yes make films 60 20 150 Highly viscous, yes not processable
[0350] As summarized in Table 1, the gel formation is good until 200 L buffer, but only gels made with 150 L and 200 L are good enough to make films by using a doctor blading technique. The next step was to determine the lowest amount of PEO. As shown in Table 2, 5-10 mg of PEO is the best amount to obtain a useful gel. As a summary, the PEO:TMPE mass ratios of 1:6 and 1:12 with a certain amount of water (150 L) were found to be the best conditions for further processing.
[0351] Further experimental results on gel formation and rubber-like material formation using various non-aqueous solvents are summarized in the following Table 3 (DMSO=dimethyl sulfoxide; DCB=dichlorobenzene; THF=tetrahydrofuran):
TABLE-US-00003 TABLE 3 TMPE PEO Volume Gel Rubber (mg) (mg) (L) formation formation polar protic EtOH 60 5 50 too liquid, immiscible 60 5 150 too liquid, immiscible isopropanol 60 5 50 too liquid, immiscible 60 5 150 too liquid, immiscible aprotic acetonitrile 60 5 50 too liquid, immiscible 60 5 150 good possible DMSO 60 5 50 too liquid, immiscible 60 5 150 too liquid, immiscible apolar DCB 60 5 50 too liquid, immiscible 60 5 150 too liquid, immiscible THF 60 5 50 too liquid, immiscible 60 5 150 too liquid, immiscible
Example 3
Application of the Rubber-Like Materials Containing Proteins Immobilized therein as Down-Converting Encapsulating Systems
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Example 4
Application of the Rubber-Like Materials Containing Proteins Immobilized therein for Diagnostic Purposes
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Example 5
Storage Stability and Thermal Stability of the Rubber-Like Materials Containing Proteins Immobilized Therein
[0354] The stability of the rubber-like materials containing proteins immobilized therein (see Example 1) was investigated under ambient conditionsi.e., storage stabilityand heating steps from room temperature to 90 C. with 10 C. steps each for 20 minutes in airi.e., thermal stability. To this end, the absorption features of the two sets of experiments were monitored over time. As shown in
Example 6
Activity Measurements of Enzymes Immobilized in the Rubber-Like Material
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[0356] Invertase Assay
[0357] The buffer for the measurement contains the substrate (sucrose, 10 mM), ATP (2 mM), NAD (1 mM), Hepes/KOH pH 7.6 (100 mM), MgCl.sub.2 (10 mM), hexokinase (1 U), phosphoglucoisomerase (PGI, 1 U, not shown) and glucose-6-phosphate dehydrogenase (G6P-DH, 1 U). When the invertase is active, it hydrolyzes sucrose into glucose and fructose. These hexoses can then be phosphorylated in an ATP-dependent manner by hexokinase. Glucose-6-phosphate is further oxidized by the enzyme glucose-6-phosphate dehydrogenase thereby producing NADH. As the absorption maximum of NADH and NAD.sup.+ differ, NADH can be exclusively detected at 340 nm. The pathway for fructose is not shown as it is the same as for glucose. The phospoglucoisomerase in the buffer converts fructose-6-phosphate into glucose-6-phosphate.
[0358] Hexokinase Assay
[0359] The buffer for the measurement contains the substrate (glucose, 5 mM), ATP (2 mM), NAD (1 mM), Hepes/KOH pH 7.6 (100 mM), MgCl.sub.2 (10 mM) and glucose-6-phosphate dehydrogenase (G6P-DH, 1 U). If the hexokinase is active the substrate glucose is phosphorylated. Glucose-6-phosphate is converted into 6-phosphogluconolacton and NAD.sup.+ is reduced in parallel. As the absorption maximum of NADH and NAD.sup.+ differ, NADH can be exclusively detected at 340 nm.
[0360] Phosphoducoisomerase (PGI) Assay
[0361] The buffer for the measurement contains the substrate (fructose-6-phosphate, 5 mM), NAD (1 mM), Hepes/KOH pH 7.6 (100 mM), MgCl.sub.2 (10 mM) and glucose-6-phosphate dehydrogenase (G6P-DH, 1 U). If the PGI is active, fructose-6-phosphate is isomerized into glucose-6-phosphate, which then can be oxidized in an NADH producing reaction. As the absorption maximum of NADH and NAD.sup.+ differ, NADH can be exclusively detected at 340 nm.
[0362] In the assays described above, the presence of the luminescent features of the NADH shows that the activity of the tested enzymes is retained when they are immobilized in a rubber-like material according to the present invention. This clearly indicates their possible application into detection kits for diagnostics.
Example 7
Application of the Rubber-Like Material Containing a Protein Immobilized therein in Hybrid White Light-Emitting Diodes (HLEDs)
[0363] In this example, a novel approach to fabricate bio-inspired hybrid white light-emitting diodes (white bio-HLEDs) combining UV- and blue-LEDs with a novel coating system using blue, green, and red fluorescent protein-based rubber materials according to the present invention is described. Three aspects constitute the main achievements of this work. Firstly, it has been demonstrated how fluorescent proteins can be used as novel down-converting materials, fulfilling the necessary requirements for this purpose, namely eco-friendly and low-cost production, easy color tunability with moderate fluorescence quantum yields, and large absorption extinction coefficients (Shcherbakova D M et al., Curr Opin Chem Biol. 2014, 20, 60; Chudakov D M et al., Physiol Rev. 2010, 90, 1103). The limitations are the need of aqueous buffer solutions, which prohibits standard coating techniques, and their moderate stability in solution under ambient conditions and/or moderate temperatures. Here, the second achievement sets in. To circumvent these problems, a new coating protocol has been developed that allows an easy-to-do homogenous covering of any kind of substrates, bringing fluorescent proteins closer to optoelectronic applications. This was possible by designing a sealing-free protein-based gel that transforms into a rubber-like material under moderate vacuum conditions. More importantly, the proteins embedded in both, gel and rubber materials, stay non-denatured for surprisingly long periods of time under ambient conditions. Thirdly, the major benefit of using a rubber material for encapsulation is the easy fabrication of a cascade architecture with a bottom-up energy transfer process (see
[0364] The blue (mTagBFP), green (eGFP), and red (mCherry) fluorescent proteins and corresponding gels were prepared as described in Example 1. It is postulated that the gel provides an excellent media in terms of rigidity and moisture to preserve the protein folding. The refraction index of the gels was also determined. Independently of the type of protein, all gels showed an average refractive index of 1.43-1.44. This value is close to the ideal one for encapsulation materials used in LEDs like silicone (Ma M et al., Opt express, 2011, 19,
[0365] A1135).
[0366] Although the gels show an excellent viscosity that allows the preparation of soft-films onto glass slides by means of doctor-blading technique, these films are not suitable for encapsulation purposes. However, the hardness of the films can be easily improved by partially drying them in a vacuum station, as described in Example 1. During this process, a water loss of around 1.5 wt. % is noted, leading to hard-films featuring mechanical properties that permit to describe them as rubber-like materials. For instance, the films are easily piled off from any substrate and even can be stretched and crumpled to obtain, for instance, a ball that keeps the luminescent features (see
[0367] Encouraged by these findings, white bio-HLEDs were fabricated combining UV- and blue-LEDsmaxima at 390 and 450 nm, respectivelywith the coating system based on the preparation of blue, green, and red emitting protein-based rubbers. For reference purposes, the LEDs were firstly modified with coatings of different thicknesses lacking the proteins. As an example, the coated blue-LED was driven at different driving currents featuring no change in the electroluminescence (EL) spectra, while the luminous efficiency value is enhanced of around 20% with a coating thickness of up to 500 m (see
[0368] Next, the UV- and blue-LEDs were modified with a single blue, green, and red protein-based coating. As expected from the excitation features of the fluorescent proteins (see
[0369] After a careful optimization, white bio-HLEDs with the architectures UV-LED/mTagBFP/eGFP/mCherry (referred to as architecture 1 in the following) and Blue-LED/eGFP/mCherry (referred to as architecture 2) were successfully prepared and analyzed. In particular, upon increasing the driven current (see
[0370] Besides the excellent color quality due to the shape of the EL spectrum, the stability of the bio-HLEDs is also sound.
[0371] In conclusion, the present invention provides the first bio-HLEDs featuring a protein-based cascade coating, which allows a perfect covering of the whole visible spectrum with a loss of less than 10% in luminous efficiency over 100 h. This has been achieved by developing a new technique to stabilize and to process fluorescent proteins in a gel that after a drying process under gentle vacuum conditions becomes a rubber material that is suitable for coating purposes. Here, it has been demonstrated that the synergy of the excellent features of the fluorescent proteinsi.e., excellent storage stability and complementary absorption-emission featureswith the easy processability of the rubber material can be exploited for designing a cascade coating suitable for lighting applications. This is the first example in which a cascade coating has been applied into HLEDs. Overall, this work opens up a new route to exploit fluorescent proteins in optoelectronic applications, and particularly in lighting applications with HLEDs.
[0372] Further details on the fabrication and characterization of the above-described bio-HLEDs are provided in the following: The UV- and blue-LED were purchased from Roschwege GmbH and Luxeon, respectively. The preparation of the bio-HLEDs concerns a two steps procedure. Firstly, the proteins-based gels (see Example 1) are deposited onto the LED wetting completely the surface. Secondly, the coated LED is transferred to the vacuum chamber under 1-10 mbar for less than 1 h. This procedure is repeated to enhance the light down-conversion efficiency of the HLED. As well, the design of a cascade coating is easily performed by repeating the above-described steps depositing subsequently blue, green, and red gels. Independently of the thickness of the coating, it can be easily piled off from the LED surface for a further analysis. The optimized thickness of the coating for devices with the architectures 1 and 2 is close to 1-1.5 mm, respectively. The bio-HLEDs were characterized by using a Keithley 2400 as a current source, while the luminous efficiency and changes of the electroluminescence spectrum were monitored by using Avantes spectrophotometer (Avaspec-ULS2048L-USB2) in conjunction with a sphere Avasphere 30-Irrad.
Example 8
Application of the Rubber-Like Material Containing a Protein Immobilized therein in a Bioreactor
[0373] The general concept is to transform a reactant into the desired product by passing the reactant solution through the rubber-like material, which contains an enzyme as the active component, by means of a vacuum system, as illustrated in
[0374] The example was performed with phosphoglucoisomerase (PGI) enzyme, the reactant NAD (featureless emission), and the product NADH (blue emitting material). Since the rubber-like material gets dissolves into the reactant solution within 5-10 min, a moderate vacuum of 100-150 mbar needs to be applied and a low amount of the reactant solution is used. Secondly, a strong vacuum of around 10-30 mbar is applied to dry and then recover the rubber-like material. Using this procedure, around 20 mL of the reactant solution has been converted into the product.
[0375] This example demonstrates that the rubber-like material according to the invention, containing an enzyme immobilized therein, can advantageously be used in a bioreactor.
Example 9
Preparation and Applications of Rubber-Like Materials Containing Non-Protein Active Materials Immobilized Therein
[0376] In this example, an easy-to-do protocol for preparing luminescent rubber-like materials based on a wide palette of active compounds, such as small-molecules, quantum dots, polymers, and coordination complexes is exemplified. The combination of this protocol with that for preparing similar rubbers based on fluorescent proteins states the universal character of this approach. This is further assessed by using comprehensive spectroscopic and rheological investigations. Furthermore, the novel luminescent rubbers are applied as down-converting packing systems to develop white hybrid light-emitting diodes (WHLEDs), which are heralded as a solid alternative to achieve energy-saving, solid-state, and white-emitting sources. As such, this work also provides a clear prospect of this emerging lighting technology by means of a direct comparison among WHLEDs fabricated with all the above-mentioned down-converting systems. Here, the use of rubbers based on coordination complexes outperforms the others in terms of both luminous efficiency and color quality with an unprecedented stability superior to 1,000 h under continuous operation conditions. This represents an order of magnitude enhancement compared to the state-of-the-art WHLEDs, while keeping luminous efficiencies of around 100 Im/W.
[0377] Introduction
[0378] The development of efficient and stable white solid-state lighting sources is one of the key technological research forefronts, as incandescent light bulbs and fluorescent lamps have reached their limit in terms of balancing luminous efficiency, stability, and environmental/recycling issues..sup.1 Two main alternatives are almost ready-to-go towards the next generation of sustainable bulbs. On one hand, organic light-emitting diodes (OLEDs) are a potential technology to provide flexible and thin lighting sources for screens and in-door luminaires..sup.2 However, despite efforts during the last 20 years, white OLEDs still show a clear trade-off in terms of low-cost production and high performance..sup.2 On the other hand, inorganic white light-emitting diodes (WLEDs) have been strongly developed by both industry and scientific communities since the pioneering works on blue-LEDs by Akasaki, Amano, and Nakamura et al. in the early 90s..sup.3 Generally speaking, the chip of the blue- or UV-emitting LEDs is coated with inorganic down-converting phosphors based on rare-earth elements like the archetypal Y.sub.3Al.sub.5O.sub.12; YAG:Ce and its derivatives..sup.4 As a result, the combination of the LED emission and that of the down-converting coating leads to WLEDs featuring high luminous efficiencies and stabilities when the packing system is optimized. Their main drawbacks are i) the high production cost due to the use of rare Earth crust materials, ii) the scarcity of efficient deep-red emitters that compromises the color quality of the white emission, and iii) the lack of efficient protocols to recycle these materials. Thus, this strategy barely addresses the basis of green economics in terms of ecological sustainability in concert with a low-cost production..sup.1
[0379] As an alternative, recent research has explored the possibility of using eco-friendly organic down-converting materials in the so-called white hybrid inorganic/organic LEDs (WHLEDs)..sup.5-8 Similar to WLEDs, the architecture of WHLEDs consists of a standard inorganic blue- or UV-LED, in which the encapsulation system is replaced by an organic-based down-converting material, which upon continuous excitation features a broad low-energy emission band (see
[0380] Up to date, there are four different approaches to develop down-converting coatings. Firstly, thin films, which consist of a mixture of organic materials with UV- or thermal-curable sealing reagents, are typically deposited either onto a glass substrate or on top of the packing of the LED (see
[0381] As the most remarkable result, the use of coordination complexes stands up among the others, featuring unprecedented stabilities of more than 1,000 h with a slight loss of luminous efficiency and no color degradation. The latter is further extrapolated to around 4,000 h, representing more than one order of magnitude enhancement in stability compared to the state-of-the-art WHLEDs. Hence, it is concluded that the combination of high luminous efficiency (100 Im/W) and stabilities of thousands of hours highlight the versatility and potentiality of the approach according to the invention for the development of WHLEDs for low- and mid-power applications.
[0382] Materials and Methods
[0383] 1. Preparation and Characterization of the Gel- and Rubber-Like Materials
[0384] All the luminescent compounds, such as small-molecules, polymers, and coordination complexes were purchased from Merck and Sigma Aldrich and used as received. The carbon quantum dots were prepared according to literature..sup.10 The gels were prepared as follows. As a first step, the branched and linear poly(ethylene oxide) compoundsi.e., trimethylolpropane ethoxylate (TMPE) with Mn. of 450 mol. wt. and linear poly(ethylene oxide) (l-PEO) with Mn. of 510.sup.6 mol. wt. and 1 mg of the luminescent compounds were mixed with different amounts of solventi.e., water or acetonitrile. Upon strong stirring (750 or 1500 rpm) under ambient conditions over night, this mixture becomes a gel. The mass ratio is optimized for the formation of a gel-like material that allows the further film forming via doctor-blading onto any kind of substrate like, for example, glass slides. The doctor-blading was performed using a rectangular stamp of a thickness of 50 m that was placed onto the support. They can also be applied onto 3D substrates by introducing them into the gels. Subsequently, the films or coated materials were introduced into a vacuum station under 1-10 mbar for less than 1 h. The final materials are best described as rubbers, which are easily peeled off from the substrate with a tweezer and can be transferred to another substrate. The thickness of the rubbers can be controlled either by the thickness of the stamp or by the subsequent deposition of one layer on top of another with an excellent adhesion. The thickness and roughness were measured using a profilometer Dektak XT from Bruker. The presence of the luminescent materials was corroborated by spectroscopic techniquessteady-state absorption and photoluminescence characterizations, as well as excited-state lifetimes and photoluminescence quantum yields that were performed by using Perkin Elmer Lambda, Fluoromax-P-spectrometer (Horiba-JobinYvon), and SPEX Fluorolog-3 (Horiba-JobinYvon) supplied with an integrated TCSPC software. The refraction index was measured by using Krss refractometer equipment from A Kross Optronic.
[0385] The rheological measurements of the gels and of the bare rubbers were carried out with an MCR 301 rheometer from Anton Paar at a temperature of 295.16 K. The gels were studied using a cone-and-plate geometry with a diameter of 25 mm and a cone angle of 1. The oscillatory measurements of the rubbers were performed with a parallel-disk configuration with a plate diameter of 25 mm and a gap width of 1 mm. Amplitude sweeps were carried out at an angular frequency of 1 rad/s in a deformation ranged between 0.1% and 2% to determine the linear viscoelastic regime of the materials studied. Frequency sweeps were carried out in the linear viscoelastic regime at angular frequencies ranging from 0.1 to 100 rad/s. The study of the impact of luminescent materials on the rheological properties of the rubbers was performed with a narrow-gap rheometer in the parallel-disk configuration at a temperature of 297.76 K. It is based on a UDS 200 rotational rheometer from Physica. As disks, it uses glass plates of 75 mm and 50 mm diameter with an evenness of /4 and /10, respectively. The gap width is set up and measured independently from the rheometer with a confocal interferometric sensor resulting in a gap width with a precision of up to 0.7 m. Further details about this setup and its alignment are provided in H. Dakhil et al., Appl. Rheol. 2014, 24, 63795. The samples were squeezed at normal forces of about 5-9 N to a gap width of 200 m.
[0386] 2. Fabrication and characterization of the WHLEDs The blue-LEDs were purchased from Luxeon (LXHL-PRO3) and Winger (WEPRB3-S1). The preparation of the WHLEDs concerns a two-step procedure. Firstly, the gels are deposited onto the LED wetting the complete surface. Secondly, the coated LED is transferred to the vacuum chamber under 1-10 mbar for less than 1 h. This procedure is repeated to enhance the light down-conversion efficiency of the WHLED. As well, the design of a cascade coating is easily performed by repeating the above-described steps depositing subsequently high- and low-energy emitting gels. Independently of the thickness of the coating, it can be easily peeled off from the LED surface for a further analysis. The optimized thickness of the coatings is mentioned further below. The WHLEDs were characterized by using a Keithley 2400 as a current source, while the luminous efficiency and changes of the electroluminescence spectrum were monitored by using Avantes spectrophotometer (Avaspec-ULS2048L-USB2) in conjunction with a sphere Avasphere 30-Irrad.
[0387] Results and Discussion
[0388] As previously reported for bio-WHLEDs, the composition of the matrixi.e., as shown in
TABLE-US-00004 TABLE 4 Test of the formation of the gel and rubber materials by changing different parameters like the nature of the solvents, the amount of the solvents, the b-PEO:I-PEO mass ratio, and the stirring conditions. Volume b-PEO/I-PEO Stirring Gel Rubber Solvent [L] [wt.] [rpm] formation formation polar protic Water 50 6:1/12:1 750/1500 Highly viscous Yes 150 6:1/12:1 750/1500 Good viscosity Yes Ethanol 50 6:1/12:1 750/1500 Immiscible No 150 6:1/12:1 750/1500 Immiscible No Isopropanol 50 6:1/12:1 750/1500 Immiscible No 150 6:1/12:1 750/1500 Immiscible No polar aprotic Acetonitrile 50 6:1/12:1 750/1500 Low viscosity No 150 6:1/12:1 750/1500 Good viscosity Yes Cyclohexanone 50 6:1/12:1 750/1500 Immiscible No 150 6:1/12:1 750/1500 Immiscible No THF 50 6:1/12:1 750/1500 Immiscible No 150 6:1/12:1 750/1500 Immiscible No apolar Toluene 50 6:1/12:1 750/1500 Immiscible No 150 6:1/12:1 750/1500 Immiscible No Hexane 50 6:1/12:1 750/1500 Immiscible No 150 6:1/12:1 750/1500 Immiscible No Chloroform 50 6:1/12:1 750/1500 Immiscible No 150 6:1/12:1 750/1500 Immiscible No
[0389] The linear viscoelastic regime of both rubbers is restricted to less than 1% strain. Here, G is always larger than G, but of the same order of magnitude. Frequency sweeps in the linear viscoelastic range revealed only a small frequency dependence, as it is typical for rubber-like materials. In general, the increase of the I-PEO content leads to an enhancement of G and G values (see
[0390] Next, the possibility was exploited to use both water and acetonitrile solvents to integrate a wide palette of luminescent materials into the rubbers. To this end, commercial luminescent materials with different emission wavelength (.sub.em) were selected, as described above, namely i) small-molecules like Coumarin 334 (1, .sub.em=496 nm), Fluorescein 27 (2, .sub.em=544 nm), zinc-tetraphenylporphyrin (ZnTPP) (3, .sub.em=609, 624, 652 nm), ii) water soluble yellowish orange emitting carbon-based quantum dots (4, .sub.em=450 nm (.sub.exc=310 nm); .sub.em=519 nm (.sub.exc=390 nm); 549 nm (.sub.exc=450 nm ,.sup.10 iii) an emitting polymer like Super Yellow (5, .sub.em=550 nm), and iv) coordination complexes such as [Ir(ppy).sub.2(acac)] (6, .sub.em=470, 490 nm) and [Ir(ppy).sub.2(tb-bpy)][PF.sub.6] (.sup.7, em=570 nm) where ppy is 2-phenylpyridine, acac is acetylacetone, and tb-bpy is 4,4-di-tert-butyl-2,2-dipyridyl. Their chemical structures are shown in
[0391] The gels were formed by mixing b-PEO:I-PEO in a ratio of 6:1 wt. and 1 mg of each luminescent materials with 150 L of acetonitrile for 1-3/5-7 and water for 4.
TABLE-US-00005 TABLE 5 Photophysical properties of 1-7 in solution and rubbers. Lifetimes.sub.sol/rubber PLQY.sub.sol/rubber [ns] Compound [%] .sub.1 .sub.2 1 68/30 0.70/0.48 3.11/1.11 2 87/36 0.97/0.24 3 3.3/1.1 1.28/0.32 1.95/1.05 4 20/5 2.60/1.64 10.30/8.27 5 69/74 0.70/1.0 0.22/0.27 6 3.9/15.7 103/651 7 7/35 65/383
[0392] Finally, a direct comparison of the stability of the luminescent compounds between the solutions and the rubbers under UV irradiation is shown in
[0393] Taking these findings into account, we fabricated white-emitting HLEDs combining a blue-LEDi.e., maximum 450 nmwith a cascade coating system combining rubbers with small-molecules (SM-WHLEDs), quantum dots (QDs-WHLEDs), polymers (P-WHLEDs), and coordination complexes (CC-WHLEDs), as described in the materials and methods section above. The preparation of the WHLEDs concerns a two-step procedure. Firstly, the gels are deposited onto the LED wetting the complete surface. Secondly, the coated LED is transferred to the vacuum chamber under 1-10 mbar for less than 1 h. This procedure is repeated to enhance the light down-conversion efficiency of the WHLED. Independently of the thickness of the coating, it can be easily peeled off from the LED surface for a further analysis. Noteworthy, the optimization of the thickness of the luminescent down-converting coatings was realized to obtain the right balance between white color quality and high luminous efficiency as shown in
[0394]
[0395] SM-WHLEDs feature an architecture of a blue-LED with a top coating based on 1 (0.14 mm)/2 (0.06 mm)/3 (0.10 mm). Upon increasing the driving current from 10 to 250 mA, the electroluminescence spectra clearly show distinguishable peaks for all small-molecules with a stable white color with, for example, CIE coordinates of 0.35/0.35 to 0.28/0.28, CRI values of 93 and 78, and CCT of 4,776 and 10,851 K for 10 and 250 mA, respectively (see
[0396] Not being able to obtain stable white lighting sources with small-molecules, we turned to investigate the QDs-WHLEDs with blue-LED/4 (0.1 mm). Similar to the SM-WHLEDs, white devices were easily achieved independently of the applied driving currents (see
[0397] Although the changes of the photoluminescence behavior of this type of materials under different excitations and environmental conditionsi.e., temperature, pH, irradiation, etc.is still under debate,.sup.10 this might be related either to interactions of the outer substituents with matrix that promote emission from trapping states or to a release of the peripheral substituents changing the core of the QDs. At this point, the QD-WHLED was probed under ambient conditions.
[0398] During the first 30 h, the electroluminescence spectra quickly evolved until a more balanced contribution in the yellow and red parts (see
[0399] Next, the use of well-known emitting polymers was investigated for the development of P-WHLEDs. The optimized device was a blue-LED/5 (0.1 mm), which independently of the applied current shows a broad electroluminescence spectrum with two maxima at 450 and 560 nm, corresponding to the blue-LED and the polymer, respectively (see
[0400] Finally, the CC-WHLEDs with the optimized architecture blue-LED/6 (0.1 mm)/7 (0.1 mm) were probed (see
[0401] Conclusions
[0402] This example provides two major thrusts in the field of WHLEDs. On one hand, the ease of preparation and application of luminescent rubbers for down-converting lighting schemes has been demonstrated. Here, important assets of the present approach are i) the in-situ preparation of the rubbers without using any cross-linking and UV- or thermal-curing methods, and ii) its versatility in terms of using any kind of luminescent materials, such as fluorescent proteins,.sup.8 small-molecules, carbon quantum dots, polymers, and coordination complexes. Here, it has been ensured that the amount of the compounds is kept constant, but a further enhancement of the luminous efficiency should be possible if the concentration of the compounds is increased, as it will reduce the coating thickness. In this regard, the latter has been optimized to obtain a high quality white emission as shown in
[0403] It is important to point out that although all-inorganic white LEDs feature much higher stabilities than the WHLEDs, the CC-WHLED provided herein shows similar CRI and luminous efficiencies to those of all-inorganic white LEDs, while its stability represents a one order of magnitude enhancement compared to the state-of-the-art hybrid white LEDs. As such, it is strongly believed that the present work constitutes a landmark for future breakthroughs in the field of WHLEDs. In this regard, a future challenge is the development of down-converting encapsulation systems for high-powerful LED arrays, which hold high operation temperatures, and in particular thermally stable organic-based coatings.
REFERENCES
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[0407] Laser Diode (Springer, 1997). [0408] 4 a) J. K. Park et al., Appl. Phys. Lett. 2004, 84, 1647; b) P. Pust et al., Nature Materials, 2014, 13, 891; c) T. M. Tolhurst et al., Adv. Opt. Mater. 2015, 3, 546; d) R.-J. Xie et al., Nitride Phosphors and Solid-State Lighting; CRC Press, 2011; e) C. Che et al., J. Phys. Chem. Lett. 2011, 2, 1268; f) R. Zhang et al., Laser Photon. Rev. 2014, 8, 158. [0409] 5 a) G. Heliotis et al., Appl. Phys. Lett. 2005, 87, 103505; b) G. Heliotis et al., Adv. Mater. 2006, 18, 334; c) E. Gu et al., Appl. Phys. Lett. 2007, 90, 031116; d) I. O. Huyal et al., J. Mater. Chem. 2008, 18, 3568; e) O. Kim et al., ACS Nano 2010, 4, 3397; f) M. Stupca et al., J. Appl. Phys. 2012, 112, 074313; g) D. Ban et al., Phys. Status Solidi 2012, 9, 2594; h) W.-S. Song et al., Chem. Mater. 2012, 24, 1961; i) E.-P. Jang et al., Nanotechnology 2013, 24, 045607; j) N. J. Findlay et al., J. Mater. Chem. C 2013, 1, 2249; k) C.-F. Lai et al., Opt. Lett. 2013, 38, 4082; I) D. D. Martino et al., Sci Rep. 2014, 4, 4400; m) P.-C. Shen et al., Sci. Rep. 2014, 4, 5307; n) J. Chen et al., J. Mater. Sci. 2014, 49, 7391; o) N. J. Findlay et al., Adv. Mater. 2014, 26, 7290; p) C. Sun et al., Nanoscale 2015, 7, 12045; q) Y. Hyein et al., Nanoscale 2015, 7, 12860. [0410] 6 a) C.-Y. Sun et al., Nat. Commun. 2013, 4, 2717; b) Q. Gong et al., J. Am. Chem. Soc. 2014, 136, 16724; c) Y. Lu et al., Chem. Commun. 2014, 50, 15443; d) Y. Cui et al., Adv. Funct. Mater. 2015, 25, 4796. [0411] 7 a) H. Tetsuka et al., J. Mater. Chem. C 2015, 3, 3536; b) D. Zhou et al., ACS Appl. Mater. Interfaces 2015, 7, 15830. [0412] 8 M. D. Weber et al., Adv. Mater. 2015, 27, 5493. [0413] 9 M. Ma et al., Opt. Express 2011, 19, A11352011. [0414] 10V. Strauss et al., J. Am. Chem. Soc. 2014, 136, 17308. [0415] 11 a) Z. Yu et al., Sci. China Chem. 2013, 56, 1075; b) J. Fang et al., Adv. Fund. Mater. 2009, 19, 2671; c) A. Asadpoordarvish et al., Adv. Eng. Mater. DOI: 10.100.sup.2/adem.201500245. [0416] 12 R. D. Costa et al., lnorg. Chem. 2011, 50, 7229.
Example 10
Preparation of Rubber-Like Materials Using TMPE as Branched Polymer and Several Linear Polymers
[0417] To demonstrate the versatility of the approach provided herein, the preparation of the gel and the final rubber-like material according to the present invention is demonstrated by using different mass ratios of the branched polymer (in this case, trimethylolpropane ethoxylate (TMPE) with a M.sub.n of 450 Da) and different linear polymers (in this case, poly(ethylene oxide) (PEO) with a M.sub.n ranging from 5000 to 8000 kDa, as well as poly(2-ethyl-2-oxazoline) (PEOx) with a M.sub.n of 500 kDa). As shown in Tables 6-8 below, several amounts of buffer solution (as otherwise used for the proteins) were added. Moreover, Table 9 summarizes the preparation of the gel and the final rubber-like material according to the invention demonstrated using different mass ratios of the branched polymer (in this case, trimethylolpropane ethoxylate (TMPE) with a M.sub.n of 450 Da) and the linear polymer (in this case, poly(ethylene oxide) (PEO) with a M.sub.n of 5000 kDa) with an aqueous saturated PEDOT:PSS solution. The rubber formation is performed as described in Example 1.
##STR00006##
TABLE-US-00006 TABLE 6 Preparation of rubber-like materials using TMPE as branched polymer and I-PEO with a M.sub.n of 5000 kDa as linear polymer TMPE PEO (mg) Water-based Gel Rubber (mg) 5000 kDa buffer (L) formation formation 60 1 150 Low viscosity to make films 60 5 150 Good viscosity to yes make films 60 10 150 Good viscosity to yes make films 60 20 150 Highly viscous, yes not processable
TABLE-US-00007 TABLE 7 Preparation of rubber-like materials using TMPE as branched polymer and I-PEO with a M.sub.n of 8000 kDa as linear polymer TMPE PEO (mg) Water-based Gel Rubber (mg) 8000 kDa buffer (L) formation formation 60 1 150 Low viscosity to yes make films 60 5 150 Good viscosity to yes make films 60 10 150 Highly viscous, yes but processable 60 20 150 Highly viscous, yes not processable
TABLE-US-00008 TABLE 8 Preparation of rubber-like materials using TMPE as branched polymer and I-PEOx with a M.sub.n of 500 kDa as linear polymer TMPE PEOx (mg) Water-based Gel Rubber (mg) 500 kDa buffer (L) formation formation 60 10 150 Low viscosity to make films 60 20 150 Good viscosity to yes make films 60 60 150 Good viscosity to yes make films
TABLE-US-00009 TABLE 9 Preparation of rubber-like materials using TMPE as branched polymer and I-PEO with a M.sub.n of 5000 kDa as linear polymer in combination with a PEDOT:PSS solution TMPE PEO (mg) Water-based Gel Rubber (mg) 5000 kDa PEDOT:PSS (L) formation formation 60 1 150 Low viscosity to make films 60 5 150 Good viscosity to yes make films 60 10 150 Good viscosity to yes make films 60 20 150 Highly viscous, yes not processable
[0418] To determine the compatibility of the fluorescent proteins embedded into the new rubbers, the same experiments were carried out using water-based buffer solution containing a fluorescent protein (0.15 mg)in this case mCherryfor the rubber formation as described for Example 1. A direct comparison of the luminescent features of the protein-based rubbers with those of the initial water-based buffer solutions indicates that there is no drastic denaturation or degradation of the protein during the rubber formation (see
Example 11
Preparation of Rubber-Like Materials Using PEI as Branched Polymer and Several Linear Polymers
[0419] Similar to example 10, this example shows the formation of rubber-like materials by changing the branched TMPE polymer for branched PEI in combination with several linear polymers. The preparation of the gel and the final rubber-like material is demonstrated by using different mass ratios of the branched polymer (in this case, polyethylenimine (PEI, with an average M.sub.w of 800 Da) and different linear polymers (in this case, poly(ethylene oxide) (PEO) with an M.sub.n of 5000 and 8000 kDa, as well as poly(2-ethyl-2-oxazoline) (PEOx) with an M.sub.n of 500 kDa). As shown in Tables 9-12, several amounts of buffer solution (as otherwise used for the proteins) were added. The rubber formation is performed as described in Example 1.
##STR00007##
TABLE-US-00010 TABLE 10 Preparation of rubber-like materials using PEI as branched polymer and PEO with a M.sub.n of 5000 kDa as linear polymer PEI PEO (mg) Water-based Gel Rubber (mg) 5000 kDa Buffer (L) formation formation 60 1 150 Low viscosity to make films 60 5 150 Low viscosity to yes make films 60 10 150 Low viscosity to yes make films 60 20 150 Good viscosity to yes make films
TABLE-US-00011 TABLE 11 PEI PEO (mg) Water-based Gel Rubber (mg) 8000 kDa Buffer (L) formation formation 60 1 150 Low viscosity to make films 60 5 150 Low viscosity to yes make films 60 10 150 Good viscosity to yes make films 60 20 150 Highly viscous, yes not processable
TABLE-US-00012 TABLE 12 PEI PEOx (mg) Water-based Gel Rubber (mg) 500 kDa Buffer (L) formation formation 60 10 150 Low viscosity to make films 60 20 150 Low viscosity to yes make films 60 60 150 Good viscosity to yes make films
[0420] To determine the compatibility of the fluorescent proteins embedded into the new rubbers, the same experiments were carried out using water-based buffer solution containing a fluorescent protein (0.15 mg)in this case mCherryfor the rubber formation as described for Example 1. A direct comparison of the luminescent features of the protein-based rubbers with those of the initial water-based buffer solutions indicates that there is no drastic denaturation or degradation of the protein during the rubber formation (see
Example 12
Preparation of Rubber-Like Materials Using TMPEMED as Branched Polymer and Several Linear Polymers
[0421] To demonstrate the versatility of the approach provided herein, the preparation of the gel and the final rubber-like material according to the present invention is demonstrated by using different mass ratios of the branched polymer (in this case, trimethylolpropane ethoxylate methyl ether diacrylate (TMPEMED) with a M.sub.n of 388 Da) and different linear polymers (in this case, poly(ethylene oxide) (PEO) with a M.sub.n of 5000 and 8000 kDa, as well as poly(2-ethyl-2-oxazoline) (PEOx) with a M.sub.n of 500 kDa). As shown in Tables 13-15, several amounts of buffer solution (as otherwise used for the proteins) were added. The rubber formation is performed as described in Example 1.
##STR00008##
TABLE-US-00013 TABLE 13 Preparation of rubber-like materials using TMPEMED as branched polymer and PEO with M.sub.n of 5000 kDa as linear polymer TMPEMED PEO (mg) Water-based Gel Rubber (mg) 5000 kDa Buffer (L) formation formation 60 1 150 Low viscosity to make films 60 5 150 Good viscosity to yes make films 60 10 150 Good viscosity to yes make films 60 20 150 Highly viscous, yes not processable
TABLE-US-00014 TABLE 14 Preparation of rubber-like materials using TMPEMED as branched polymer and PEO with M.sub.n of 8000 kDa as linear polymer TMPEMED PEO (mg) Water-based Gel Rubber (mg) 8000 kDa Buffer (L) formation formation 60 1 150 Low viscosity yes to make films 60 5 150 Good viscosity yes to make films 60 10 150 Highly viscous, yes but processable 60 20 150 Highly viscous, yes not processable
TABLE-US-00015 TABLE 15 Preparation of rubber-like materials using TMPEMED as branched polymer and PEOx with M.sub.n of 500 kDa as linear polymer TMPEMED PEOx (mg) Water-based Gel Rubber (mg) 500 kDa Buffer (L) formation formation 60 10 150 Low viscosity to make films 60 20 150 Good viscosity to yes make films 60 60 150 Good viscosity to yes make films
[0422] To determine the compatibility of the fluorescent proteins embedded into the new rubbers, the same experiments were carried out using water-based buffer solution containing a fluorescent protein (0.15 mg)in this case mCherryfor the rubber formation as described for Example 1. A direct comparison of the luminescent features of the protein-based rubbers with those of the initial water-based buffer solutions indicates that there is no drastic denaturation or degradation of the protein during the rubber formation (see
Example 13
Preparation of Rubber-Like Materials Containing Different Luminescent Materials Immobilized Therein
[0423] The preparation of the gel and the rubber-like material according to the present invention is demonstrated using different mass ratios of the branched polymer (in this case, trimethylolpropane ethoxylate (TMPE) with an M.sub.n of 450 Da) and the linear polymer (in this case, poly(ethylene oxide) (PEO) with an M.sub.n of 5000 kDa), as shown in Tables 16 and 17 below. The rubber formation is performed as described in Example 1. In particular, the two polymers are mixed at different mass ratios as shown in Table 17 below. Although TMPE is a low viscous liquid, the PEO does not dissolve even at high stirring conditions. To facilitate this process, several amounts of acetonitrile or water, which is already mentioned above in Tables 1 and 2 (see Example 2), were added.
TABLE-US-00016 TABLE 16 Preparation of rubber-like materials using branched and linear polymers in a mass ratio of 12:1 and different amounts of acetonitrile TMPE PEO Acetonitrile Gel Rubber (mg) (mg) (L) formation formation 60 5 50 Highly viscous, not processable 60 5 100 Highly viscous, but processable 60 5 150 Good viscosity to yes make films 60 5 200 Low viscosity to yes make films 60 5 300 Low viscosity to yes make films 60 5 400 Low viscosity to yes make films 60 5 500 Low viscosity to yes make films
TABLE-US-00017 TABLE 17 Preparation of rubber-like materials using branched and linear polymers in different mass ratios and acetonitrile TMPE PEO Acetonitrile Gel Rubber (mg) (mg) (L) formation formation 60 1 150 Low viscosity to make films 60 5 150 Good viscosity to yes make films 60 10 150 Good viscosity to yes make films 60 20 150 Highly viscous, yes not processable
[0424] As summarized in Table 16, the gel formation is the best with 150 L acetonitrile, which indicates that they are good enough to make films by using a doctor blading technique. The next step was to determine the lowest amount of PEO. As shown in Table 17, 5-10 mg of PEO is the best amount to obtain a useful gel. As a summary, the PEO:TMPE mass ratios of 1:6 and 1:12 with a certain amount of water (150 L) were found to be the best conditions for further processing.
[0425] The final layer is best described as rubber-like material in which the loss of most of the acetonitrile provokes the collapse of the network structure. Notably, the solvent is not recovered over weeks under ambient storage conditions (see
[0426] Preparation of Rubber-Like Materials Containing Different Luminescent Compounds Embedded Therein
[0427] The formation of the luminescent gels and rubber-like materials is carried out in a similar procedure as described in Example 1. Here, commercially available luminescent compounds can be added as powder directly to the mixture of branched and linear poly(ethylene oxide) compoundsi.e., trimethylolpropane ethoxylate (TMPE) with a M.sub.n of 450 Da and linear poly(ethylene oxide) (l-PEO) with M.sub.n of 510.sup.6 Da, with a mass ratio of 6:1, respectively, while the solventi.e., either water or acetonitrile, depending on the properties (solubility, polarity, etc.) of the luminescent compoundsis added subsequently. Here, it is also proposed that the terminal hydroxyl groups provide a high compatibility with the acetonitrile solution, retaining enough solvent molecules within network. The gel network is mainly provided by the TMPE, while the I-PEO acts as a gelation agent. In the studied mass range of luminescent materials, the formation of the gel and the final rubber material are independent of the amount of embedded compounds. Furthermore, the luminescent gels and rubber-like materials exhibit similar properties as the above mentioned protein-based ones described in Examples 1 and 2 i.e., applicable onto 3D substrates by introducing them into the gels; the thickness of the rubber-like materials can be controlled either by the thickness of the stamp or by the subsequent deposition of one layer on top of another with excellent adhesion. The presence of the luminescent compounds was corroborated by steady-state spectroscopic techniquessteady-state absorption and photoluminescence characterizations were performed by using Perkin Elmer Lambda and Fluoromax-P-spectrometer from HORIBA Jobin Yvon, respectively.
[0428] A direct comparison of the luminescent features of the final materials with those of the initial solutions indicates that there is no drastic degradation of the compounds during the formation of the luminescent rubber-like material; the small changesi.e., maxima shift of around 5-10 nm and slight broadening of the spectrumnoted when comparing the emission features from solution and rubber-like materials are produced by small interactions of the luminescent compounds with the surrounding matrix, which do not significantly affect the luminophore.