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(12) United States Patent (10) Patent No.: US 8,561,811 B2 Bluchel Et Al USOO8561811 B2 (12) United States Patent (10) Patent No.: US 8,561,811 B2 Bluchel et al. (45) Date of Patent: Oct. 22, 2013 (54) SUBSTRATE FOR IMMOBILIZING (56) References Cited FUNCTIONAL SUBSTANCES AND METHOD FOR PREPARING THE SAME U.S. PATENT DOCUMENTS 3,952,053 A 4, 1976 Brown, Jr. et al. (71) Applicants: Christian Gert Bluchel, Singapore 4.415,663 A 1 1/1983 Symon et al. (SG); Yanmei Wang, Singapore (SG) 4,576,928 A 3, 1986 Tani et al. 4.915,839 A 4, 1990 Marinaccio et al. (72) Inventors: Christian Gert Bluchel, Singapore 6,946,527 B2 9, 2005 Lemke et al. (SG); Yanmei Wang, Singapore (SG) FOREIGN PATENT DOCUMENTS (73) Assignee: Temasek Polytechnic, Singapore (SG) CN 101596422 A 12/2009 JP 2253813 A 10, 1990 (*) Notice: Subject to any disclaimer, the term of this JP 2258006 A 10, 1990 patent is extended or adjusted under 35 WO O2O2585 A2 1, 2002 U.S.C. 154(b) by 0 days. OTHER PUBLICATIONS (21) Appl. No.: 13/837,254 Inaternational Search Report for PCT/SG2011/000069 mailing date (22) Filed: Mar 15, 2013 of Apr. 12, 2011. Suen, Shing-Yi, et al. “Comparison of Ligand Density and Protein (65) Prior Publication Data Adsorption on Dye Affinity Membranes Using Difference Spacer Arms'. Separation Science and Technology, 35:1 (2000), pp. 69-87. US 2013/0210111A1 Aug. 15, 2013 Related U.S. Application Data Primary Examiner — Chester Barry (62) Division of application No. 13/580,055, filed as (74) Attorney, Agent, or Firm — Cantor Colburn LLP application No. PCT/SG2011/000069 on Feb. 18, 2011, now Pat. No. 8,440,441. (57) ABSTRACT A substrate having compounds disposed thereon for immo (30) Foreign Application Priority Data bilizing a functional molecule, each compound having a chain including: a moiety R that is chemically coupled to the Feb. 19, 2010 (GB) ................................... 10O2824.9 Substrate, the moiety R being selected from the group con (51) Int. Cl. sisting of an ether, ester, carbonyl, carbonate ester, thioether, BOID 39/00 (2006.01) disulfide, Sulfinyl, Sulfonyl, and carbonothioyl; and an (52) U.S. Cl. epoxide-containing moiety that is coupled to the moiety R by USPC ............ 210/500.27: 210/500.37; 210/500.29; a linker including at least one nucleophilic group. Methods of 21 Ofé06 preparing the Substrate and use of the Substrate are also dis (58) Field of Classification Search closed. USPC ................... 210/500.27,500.37,500.29, 606 See application file for complete search history. 26 Claims, 5 Drawing Sheets U.S. Patent Oct. 22, 2013 Sheet 1 of 5 US 8,561,811 B2 1 OO 110 O 112 Han- —- Step A-1 on 114 X 8), eelector —- Step A-2 or. x (6)(B) (eelfell. cle, a 116 Hal 1 \{1 — Step A-3 O R R RW f O x (8.68, wcre p (),g Y n 140 144 142 2. t 120 —- Step A-4 130 & O rxX (e.8 (8) = CuCE =9, CIA}. at h,u-Y . Z \ Fig. 1 a U.S. Patent Oct. 22, 2013 Sheet 2 of 5 US 8,561,811 B2 200 210 212 Half- - - Step Bl O / f 214 to (8,169\ m n v.cctva p. ( —- Step B2 or x (el?ef elector 216 Hal 1 \{1 os- Step B-3 R.Y. (RRY L. C 4.18 etc ichte, Y s>< N 242 1. Fig. 1b U.S. Patent Oct. 22, 2013 Sheet 3 of 5 US 8,561,811 B2 300 310 312 Hal 1 \{1 —- Step C-1 O 314 to (8), eelecte-q O one- Step C-2 orO x-(e)teelectep\ /d O 320 - - Step C-3 330 R Y (R R 2. orrxO C C-C C=CP \At / ot Fig. 2 U.S. Patent Oct. 22, 2013 Sheet 4 of 5 US 8,561,811 B2 410 400 412 Cir- —- Step D-1 414 N-N-N-N-NHe —- Step D-2 416 450 p 452 D se C" r O U.S. Patent Oct. 22, 2013 Sheet 5 of 5 US 8,561,811 B2 500 510 —- Step E-1 512 514 —- Step E-3 520 530 US 8,561,811 B2 1. 2 SUBSTRATE FOR IMMOBILIZING of the generally poorly reactive polymeric material. Surface FUNCTIONAL SUBSTANCES AND METHOD modification can be achieved by physical means, such as FOR PREPARING THE SAME non-covalent attachment of an affinity spacer, or chemical means such as glutaraldehyde activation, cyanogen bromide CROSS REFERENCE TO RELATED activation, bromoacetylation, diazotation, ionizing-radiation APPLICATIONS induced oxidation and chemical grafting. The non-covalent attachment of an affinity spacer is, how The present application is a divisional application of U.S. ever, associated with poor reproducibility and/or unstable patent application Ser. No. 13/580,055, filed on Aug. 20. binding to the surface of the substrates. Some covalent attach 2012, the entire contents of which are incorporated herein by 10 ments, most noteworthy imines, but to a lesser extent also reference. The Ser. No. 13/580,055 application is the U.S. esters, can be hydrolyzed under the reaction conditions used National Stage of Application No. PCT/SG2011/000069, for enzymatic reactions, resulting in partial loss of immobi filed Feb. 18, 2011. Priority under 35 U.S.C. S 119(a) and 35 lized enzyme and leakage of enzyme into the reaction U.S.C. S365(b) is hereby claimed from GB Application No. medium. Such problems may affect, amongst others, immo 1002824.9, filed Feb. 19, 2010, the disclosures of which are 15 bilization methods based on glutaraldehyde activation and both also incorporated herein by reference. bromoacetylation. Whilst diazotation, cyanogen bromide activation, ionizing-radiation induced oxidation, and chemi TECHNICAL FIELD cal grafting produce covalent bonds which are more stable than non-covalent bonds, these methods involve the use of The present invention generally relates to a substrate for hazardous, expensive, complicated, and/or harsh reaction immobilization of functional Substances. The present inven conditions. tion also relates to methods of preparing the Substrate. Some of these methods also result in a high net charge on the Solid Support, which causes undesirable non-specific elec BACKGROUND trostatic binding of the functional and/or biological Substance 25 during Subsequent procedures in a biological/chemical pro Biological and chemical assays and processes that are used cess. Another common problem encountered with the use of analytically or preparatively for research, clinical, diagnostic harsh reaction conditions is the unfavorable modification of and industrial purposes often require fixation, or immobiliza Surface properties, which may hamper the attachment of a tion, of a functional Substance onto a solid Support (or Sub functional and/or biological Substance, particularly a large strate). This fixation often improves the stability and versa 30 polymeric Substance. This can lead to low loading of the tility of the substance without compromising its effectiveness functional and/or biological substance onto the substrate. Yet and activity, and enables repeated usage of the substance. For other problems encountered with some commercially avail example, functional Substances which include biological Sub able activated Solid Supports are low stability, pronounced stances such as enzymes, are typically immobilized on an toxicity and a lack of biocompatibility, resulting in short shelf inert Support like silica or polyacrylamide gel to improve their 35 life, difficult handling, and limited applicability for medical stability against changing pH or temperature conditions when purposes. used in enzyme-catalyzed industrial processes, and to facili Some of these methods rely on the further modification of tate their Subsequent separation from the reaction products. “pre-activated Supports with an epoxysilane coupling agent This enables re-use of the immobilized enzymes and signifi for the immobilization of hydrophilic molecules. Other meth cantly facilitates product purification, which leads to more 40 ods rely on preparation of a Substrate by reacting a bisepoxy cost-effective processes. oxirane linker to immobilize a molecule to the substrate. The Immobilization of a functional Substance including a bio aliphatic linkers used in these methods lead to a decrease in logical Substance may be effected by physical immobilization the amount of reactive groups available for immobilization, a or chemical immobilization. One form of physical immobi decrease in biocompatibility and a decrease in reproducibil lization is physical adsorption (physisorption), where the 45 ity. functional or biological Substance is attached to the Substrate There is a need to provide methods of preparing a substrate via encapsulation or electrostatic, hydrophobic or Van der for immobilization of functional and biological Substances Waals forces. Whilst physical adsorption provides a relatively that overcome, or at least ameliorate, one or more of the simple immobilization method with wide applicability to a disadvantages described above. whole range of functional and/or biological Substances, it 50 There is a need to provide methods that are convenient, often does not provide a sufficiently stable immobilization inexpensive, robust, and reliable for preparing a Substrate for and is susceptible to leaching of the immobilized functional immobilization of functional and biological Substances. and/or biological Substances. There is also a need to provide substrates which are stable, A more stable method of immobilization of functional easy to handle, inexpensive, non-toxic, biocompatible and and/or biological Substances is chemical immobilization, 55 bio-degradable for immobilization of functionally and bio which covalently binds the functional and/or biological sub logically active Substances, and which can be used for immo stance to the Substrate as a result of a chemical reaction. bilization of a wide range of Substances at high loading den Chemical immobilization typically results in improved activ sities with improved activity and reactivity. ity, reduced non-specific adsorption, and higher stability of the functional and/or biological substance.
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