WO2015106329A1 - Carbon nanotubes with access to macromolecule bonding restricted by an albumin coating (racnt-bsa) - Google Patents

Carbon nanotubes with access to macromolecule bonding restricted by an albumin coating (racnt-bsa) Download PDF

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Publication number
WO2015106329A1
WO2015106329A1 PCT/BR2015/000003 BR2015000003W WO2015106329A1 WO 2015106329 A1 WO2015106329 A1 WO 2015106329A1 BR 2015000003 W BR2015000003 W BR 2015000003W WO 2015106329 A1 WO2015106329 A1 WO 2015106329A1
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Prior art keywords
bsa
racnt
carbon nanotubes
restricted
albumin
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PCT/BR2015/000003
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French (fr)
Portuguese (pt)
Inventor
Eduardo Costa DE FIGUEIREDO
Adriano Francisco BARBOSA
Pedro Orival LUCCAS
Valéria Maria Pereira BARBOSA
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Universidade Federal De Alfenas-Unifal-Mg
Fundação De Amparo À Pesquisa De Minas Gerais - Fapemig
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Priority claimed from BR102014001074-2A external-priority patent/BR102014001074B1/en
Application filed by Universidade Federal De Alfenas-Unifal-Mg, Fundação De Amparo À Pesquisa De Minas Gerais - Fapemig filed Critical Universidade Federal De Alfenas-Unifal-Mg
Publication of WO2015106329A1 publication Critical patent/WO2015106329A1/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/34—Size selective separation, e.g. size exclusion chromatography, gel filtration, permeation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • B01J20/289—Phases chemically bonded to a substrate, e.g. to silica or to polymers bonded via a spacer
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3204—Inorganic carriers, supports or substrates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3214—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating
    • B01J20/3217—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond
    • B01J20/3219—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond involving a particular spacer or linking group, e.g. for attaching an active group
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/3272—Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B01J20/3274—Proteins, nucleic acids, polysaccharides, antibodies or antigens
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3291—Characterised by the shape of the carrier, the coating or the obtained coated product
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B32/00—Carbon; Compounds thereof
    • C01B32/15—Nano-sized carbon materials
    • C01B32/158—Carbon nanotubes
    • C01B32/168—After-treatment
    • C01B32/174—Derivatisation; Solubilisation; Dispersion in solvents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/20—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the sorbent material
    • B01D15/206—Packing or coating
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures

Definitions

  • the subject of the invention relates to carbon nanotubes restricted to binding of macromolecules by means of external coating with bovine serum albumin (RACNT-BSA) restricted access carbon nanotube capped bovine serum albumin.
  • Carbon nanotubes are surrounded by a layer of bovine serum albumin, chemically crosslinked, forming a protective cap.
  • RACNT-BSA bovine serum albumin
  • the proteins in the sample do not bind to the sorbent due to the protection of the outer layer of albumin.
  • Even the low molecular weight analytes can diffuse in the middle of the albumin layer and bind to the nanotubes.
  • the material developed is of synthetic origin and can be used as a solid reagent for preconcentration / selective extraction of low molecular weight analytes in protein samples, thus avoiding the need to perform prior procedures for the decomposition of the sample matrix.
  • the CNTs are cylindrical in shape like graphite sheets rolled in the shape of a straw, and may have one or several layers, diameter from 4 to 30 nm and length of 1 ⁇ .
  • the properties of the CWT the high chemical and physical stability and, especially, the high surface area, favorable conditions in adsorption processes are mentioned (Byszewski, P., Lange, H., Huczko, A., Be nke, FJ of Physics Chemistry Solide., 11 (1997) 1679).
  • the excellent adsorptive properties of the CNTs are attributed to the hexagonal arrangement of the carbon atoms in the graphite sheet, which gives strong interaction of atoms and molecules higher than those observed in activated carbon.
  • These materials are widely used in sorption processes, for the solid phase extraction of various analytes and as a chromatographic column filling. However, all applications are restricted to aqueous solutions. When the analytes are found in medium with high concentration of macromolecules (eg whole blood, plasma, serum, food samples, etc.), the material does not present selectivity and retains large amount of proteins on its external surface, causing loss of selectivity, decrease in their useful life and risk of analytical column degradation when the analysis is conducted by solid phase and chromatographic extraction techniques.
  • macromolecules eg whole blood, plasma, serum, food samples, etc.
  • RAM Restricted access materials
  • silica-based sorbents and provided with pores where the various low molecular weight molecules are capable of binding.
  • such materials have their outer surface modified with hydrophilic binders to the point of chemically making the binding of undesired macro-molecules present in the matrix chemically unviable (Sad ⁇ lek P., Satinsky D., Solich P., Anal. Chem., 26 (2007) 375- 384.).
  • RAMs have been used in the direct extraction of organic analytes in protein fluids such as blood, plasma, serum, etc. Examples of applications of restricted access materials in analytical methods have been observed for the extraction of organic compounds using chromatographic columns and pre-column chromatography.
  • RAMs were already employed in commercial chromatography columns containing silica, C18, C8 and MIP (molecular printing polymers) (EP2156182; O2008148547; US20040191537; US200281050; WO2002101362; EP135721; EP1053021; WO1999039739; PI1020120153394).
  • CNTs are highly adsorptive materials, which are not very selective and have an undesirable ability to retain macromolecules, making their direct application in the extraction of analytes in protein fluids impossible.
  • CNT capable of eliminating macromolecules there are no reports in the literature of CNT capable of eliminating macromolecules, and these materials are promising in the direct separation / extraction / preconcentration of organic and inorganic analytes into protein fluids.
  • the main object of the invention is to describe the method of obtaining and using a CNT restricted to macromolecule binding (RACNT-BSA).
  • CNTs were coated / encapsulated by a layer of chemically cross-linked serum bovine albumin.
  • Direct extraction of cadmium into human blood serum followed by atomic absorption analysis is described as a model in that patent.
  • any other inorganic or organic analytes can be extracted from protein fluids using the RACNT-BSA. To date, there are no reports in databases on the technology developed.
  • RACNT-BSA has the potential for wide use and commercialization in industries and laboratories that work with analyzes of metals and organic compounds in biological fluids, and can be packaged in any extraction system that uses solid sorbents, with the great advantage of being possible the injection directly from protein fluids without any previous deproteinization step.
  • the aim of the present invention is the development and use of carbon nanotubes restricted to binding to macromolecules by coating with albumin (RACNT-BSA), and which are capable of directly extracting analytes present in protein matrices without the prior need of decomposition of the matrix by slow procedures such as microwave decomposition, dry route, humid route, etc. These materials are capable of eliminating about 100% of the macromolecules present in protein fluids undergoing extraction.
  • the principle of elimination of the macromolecules comes from the physico-chemical nature of the sorbent, since the immobilization of the BSA on the surface of the CNT blocks the CNT binding sites preventing binding of the same to the proteins of the sample.
  • RACNT-BSA has a long shelf-life, proven by maintaining its performance even after about 300 consecutive extractions of human serum samples for cadmium analysis. In terms of cost, the production of 1 g of RACNT-BSA requires about R $ 150.00. It is noteworthy that in the RACNT-BSA proposed in this application for patent protection does not resemble the patents reported on CTN. Brief Description of the Figures
  • Figure 1 Representative scheme of RACNT-BSA synthesis. (1) CNT, (2) reagents responsible for functionalization, (3) functionalisation reaction, (4) functionalized CNTs, (5) BSA (bovine serum albumin), (6) CNT coating step, (7 and 8 ) RACNT-BSA.
  • BSA bovine serum albumin
  • RACNT-BSA single or multiple-walled CNTs can be coated either directly or after a functionalization reaction.
  • Unmodified CNTs exhibit supportive characteristics and are mainly used for extraction / preconcentration of organic compounds.
  • the functionalization of the material provides better characteristics.
  • the CNTs can be functionalized with different organic molecules using covalent or non-covalent binding strategies (Antonio V. Herrera-Herrera et al., Analytica Chimica Acta, 734 (2012) 1).
  • the main functionalisations are:
  • the reagents used are: NH 3 . H 2 O / H 2 O 2 ; H 2 SO 4 / H 2 0 2 ; HNO 3 ; KMNO 4 ; 2CH 2 0 7; HNO 3 / 3 ⁇ 40 2 .
  • This functionalization initially involves the oxidation of CNTs and subsequent reaction with amines, such as: ethylenediamine (EDA) (Z. Zang, Z. Hu, Z. Li, Q. He, X. Chang, J. Hazard, Mater, 172 (2009) 958); Tris (2 ' aminoethyl) amine (TAA) (Y. Cui, S. Liu, Z.-J. Hu, X.-H. Liu, H. W .; Gao, Microchim. Acta 174 (2011) 107); 2- aminobenzothiazole (ABT) (R. Li, X. Ang., Z. Li. 2. Zang. Z. Hu. D. Li. Z.
  • Another way of functionalizing CNTs can be obtained by the use of N 2 induced plasma.
  • This technique allows the formation of ⁇ - C * and - CN * groups in CNTs which can then react with other organic compounds and receive other functional groups.
  • This technique aims to replace the previous stage of oxidation, used in other functionalisations of the CNT, to present higher efficiency since the oxidation stage can degrade the CNT.
  • the main modifications with this technique involve the use of polyacrylamide (PAAM), poly (N, N-dimethylacrylamide) PDMA (D. Shao, X. Ren, J. Hu, Y. Chen, X. Wang, Colloids Surf., A 360 (2010) 74); (VP) (X. Ren, D. Shao, G. Zhao, G. Sheng, J.
  • the functionalized CTNs were then coated with BSA (Menezes M.L., Felix G, J. Liq. Chromatogr RT, 21 (1998) 2863; Lima VV, Cassiano NM, Cass Q.B., Qu ⁇ mica Nova, 29 (2006) 72), and it is important to emphasize that any other type of CNT functioned for being subjected to this coating process.
  • BSA Melezes M.L., Felix G, J. Liq. Chromatogr RT, 21 (1998) 2863; Lima VV, Cassiano NM, Cass Q.B., Qu ⁇ mica Nova, 29 (2006) 72
  • a volume of 1 to 50 mL of an aqueous solution of sodium borohydride of 0.05 to 5% (w / v) were percolated by the cartridge for the purpose of reducing the imines (originating from the previous reaction) to amines which are more chemically stable moieties, thereby conferring chemical stability on the material.
  • the RACNT-BSA was washed in abundance with water to remove the remaining reagents from the coating process.
  • a column of 2 to 7 mm internal diameter by 5 cm in length was filled with RACNT-BSA and coupled in a chromatographic system to the analytical column.
  • 25 ⁇ l of an aqueous BSA standard 44 mg.mL- 1 (the same human blood serum protein concentration) were injected through the column and the resulting peak (detected at 254 nm) was compared to the peak obtained from the injection of the same BSA standard on the system without the column. It was verified that there was practically no albumin retention in the RACNT-BSA (with recovery of 101 ⁇ 1%), indicating that the coating satisfactorily prevented the binding of proteins on the CNT surface.
  • the RACNT-BSA-containing column was then coupled at the position of the sampling loop on a manual switch injector employed in the flow preconcentration system, eluted straight to a heated nickel tube atomic absorption equipment (TS-FF-AAS, from thermospray flame furnace atomic absorption spectrometry).
  • TS-FF-AAS heated nickel tube atomic absorption equipment
  • the system was optimized and validated for cadmium extraction in serum and human blood plasma.
  • the method was efficient in the direct analysis (without the need of any preliminary step of sample preparation) of cadmium, with limit of quantification of 0.8 ug / L>.
  • the material besides allowing the direct extraction of cadmium, still presented high preconcentration power (preconcentration factor of 5.7).

Abstract

The present invention relates to the development of a solid sorbent constituted by carbon nanotubes with access to macromolecule bonding restricted by an albumin coating (RACNT-BSA). This material is an extractor/preconcentrator capable of eliminating 100% of the macromolecules during extraction processes with proteic fluids, thus dispensing with the decomposition of the organic matrix prior to the assays. The material is easy to produce at a low cost, and the performance in terms of selectivity and capacity of elimination of macromolecules is superior to that of commercially available sorbents. The material has a long service life (about 300 extractions) and a high commercialisation potential.

Description

NANOTUBOS DE CARBONO RESTRITOS À LIGAÇÃO COM MACROMOLÉCULAS CARBON NANOTUBES RESTRICTED TO CONNECTION WITH MACROMOLECULES
POR MEIO DE RECOBRIMENTO COM ALBUMINA (RACNT-BSA) BY ALBUMIN RECOVERY (RACNT-BSA)
Campo da Invenção Field of the Invention
[001] O objeto de invenção refere-se à nanotubos de carbono restritos à ligação de macromoléculas por meio de revestimento externo com albumina sérica bovina (RACNT-BSA - em inglês restricted access carbon nanotube capped wíth bovine serum albumín) . Nanotubos de carbono são envolvidos por uma camada de albumina sérica bovina, entrecruzada quimicamente, formando uma capa protetora. Quando uma amostra proteica é percolada pelo RACNT-BSA, as proteínas da amostra não se ligam ao sorbente devido à proteção da capa externa de albumina. Jã os analitos de baixo peso molecular conseguem se difundir no meio da capa de albumina se ligam aos nanotubos. O material desenvolvido é de origem sintética e pode ser empregado como reagente sólido para a pré-concentração/extração seletiva de analitos de baixo peso molecular em amostras proteicas, evitando assim a necessidade de realização de procedimentos prévios de decomposição da matriz da amostra.  The subject of the invention relates to carbon nanotubes restricted to binding of macromolecules by means of external coating with bovine serum albumin (RACNT-BSA) restricted access carbon nanotube capped bovine serum albumin. Carbon nanotubes are surrounded by a layer of bovine serum albumin, chemically crosslinked, forming a protective cap. When a protein sample is percolated by RACNT-BSA, the proteins in the sample do not bind to the sorbent due to the protection of the outer layer of albumin. Even the low molecular weight analytes can diffuse in the middle of the albumin layer and bind to the nanotubes. The material developed is of synthetic origin and can be used as a solid reagent for preconcentration / selective extraction of low molecular weight analytes in protein samples, thus avoiding the need to perform prior procedures for the decomposition of the sample matrix.
Fundamentos da Invenção:  Background of the Invention:
Nanotubos de Carbono {CNT)  Carbon Nanotubes (CNT)
[002] Os CNT de paredes múltiplas (1991) e os CNT com parede simples (1993) foram produzidos pela primeira vez por Sumio Iijima (S. Iijima, Nature . 354 (1991) 56). De acordo com dados de microscopia eletrônica de varredura, os CNT possuem forma cilíndrica como folhas de grafite enroladas na forma de um canudo, podendo apresentar uma ou várias camadas, diâmetro de 4 a 30 nm e comprimento de 1 μτη. [003] Dentre as propriedades dos CWT, cita-se a elevada estabilidade química e física e, principalmente, a elevada área superficial, condições propícias em processos de adsortivos (Byszewski, P., Lange, H., Huczko, A., Be nke, F. J. of Physícs Chemistry Solide., 11 (1997) 1679). As excelentes propriedades adsortivas dos CNT são atribuídas ao arranjo hexagonal dos átomos de carbono na folha de grafite, o que confere forte interação de átomos e moléculas superiores àquelas verificadas em carbono ativado. Esses materiais são amplamente empregados em processos sortivos, para extração em fase sólida de diversos analitos e como recheio de colunas cromatográfícas . Porém todas as aplicações são restritas a soluções aquosas. Quando os analitos encontram~se em meio com alta concentração de macromoléculas (e.g. sangue total, plasma, soro, amostras de alimentos, etc) , o material não apresenta seletividade e retém grande quantidade de proteínas em sua superfície externa, causando perda de seletividade, diminuição de sua vida útil e risco de degradação de colunas analíticas quando a análise ê conduzida por técnicas de extração em fase solida e cromatográficas . As patentes apresentadas a seguir relatam a síntese e funcionalização de nanotubos de carbono de paredes simples, duplas e múltiplas e foram encontradas nos bancos de dados nacionais e internacionais: US20080206125 ; WO2009020958; R1020080078879 ; WO2008100325 ; EP1940547; O2008079465; KR1020080052581 ; WO2008048313 ; O2007134175 ; US20070256929; EP1797950; WO2007046713 ; EP1747309; EP1692077; JP2006188389 ; EP1644287; US20060039849 ; WO2005106086; US20050123467 ; US20050042162 ; US20040245088 ; EP1481946; WO2003068676 ; EP1328472; WO2003048038 ;Multiple wall CNTs (1991) and single wall CNTs (1993) were first produced by Sumio Iijima (S. Iijima, Nature, 354 (1991) 56). According to scanning electron microscopy data, the CNTs are cylindrical in shape like graphite sheets rolled in the shape of a straw, and may have one or several layers, diameter from 4 to 30 nm and length of 1 μτη. Among the properties of the CWT, the high chemical and physical stability and, especially, the high surface area, favorable conditions in adsorption processes are mentioned (Byszewski, P., Lange, H., Huczko, A., Be nke, FJ of Physics Chemistry Solide., 11 (1997) 1679). The excellent adsorptive properties of the CNTs are attributed to the hexagonal arrangement of the carbon atoms in the graphite sheet, which gives strong interaction of atoms and molecules higher than those observed in activated carbon. These materials are widely used in sorption processes, for the solid phase extraction of various analytes and as a chromatographic column filling. However, all applications are restricted to aqueous solutions. When the analytes are found in medium with high concentration of macromolecules (eg whole blood, plasma, serum, food samples, etc.), the material does not present selectivity and retains large amount of proteins on its external surface, causing loss of selectivity, decrease in their useful life and risk of analytical column degradation when the analysis is conducted by solid phase and chromatographic extraction techniques. The following patents report the synthesis and functionalization of single-, double- and multiple-walled carbon nanotubes and were found in national and international databases: US20080206125; WO2009020958; R1020080078879; WO2008100325; EP1940547; O2008079465; KR1020080052581; WO2008048313; O2007134175; US20070256929; EP1797950; WO2007046713; EP1747309; EP1692077; JP2006188389; EP1644287; US20060039849; WO2005106086; US20050123467; US20050042162; US20040245088; EP1481946; WO2003068676; EP1328472; WO2003048038;
WO2003038163 ; R1020020040644 ; EP1192104 ; EP1165440 EP1061044; WO2000073205 ; O2000051936 ; WO2000017102 ; EP1973845; US20090099016 ; WO2012070527 ; US20110150746 ; WO2010147192; US08182782; US20120114550 ; US20120107610 ; US20120070566; SG177396; PI1005774-9 A2 ; WO/2012/168899 ; PI1002753-0 A2 ; PI1004450-7 A2 ; P11011517-0 A2 ; C10701709-0 E2; PI0801851-0 A2 ; PI0800605-9 A2 ; US20120237436 ; US20120234204; WO2012125528 ; EP2493811; US20120145997 ; US20120171107. WO2003038163; R1020020040644; EP1192104; EP1061044; WO2000073205; O2000051936; WO2000017102; EP1973845; US20090099016; WO2012070527; US20110150746; WO2010147192; US08182782; US20120114550; US20120107610; US20120070566; SG177396; PI1005774-9 A2; WO / 2012/168899; PI1002753-0 A2; PI1004450-7 A2; P11011517-0 A2; C10701709-0 E2; A2; PI0800605-9 A2; US20120237436; US20120234204; WO2012125528; EP2493811; US20120145997; US20120171107.
Materiais de Acesso Restrito - RAM  Restricted Access Materials - RAM
[004] Os materiais de acesso restrito (RAM) são sorventes recentemente propostos, a base de sílica, e dotados de poros onde as diversas moléculas de baixo peso molecular são capazes de se ligar. Além disso, esses materiais têm sua superfície externa modificada com ligantes hidrofílicos a ponto de inviabilizar quimicamente a ligação de macrotnoléculas indesejadas presentes na matriz (Sadílek P., Satínsky D., Solich P., Anal. Chem., 26 (2007) 375-384.) . Os RAM têm sido empregados na extração direta de analitos orgânicos em fluidos protéicos como sangue, plasma, soro, etc. Exemplos de aplicações de materiais de acesso restrito em métodos analíticos têm sido observados para a extração compostos orgânicos utilizando colunas cromatogrãficas e pré-colunas cromatogrãficas . Como reagente sólido, os RAMs jã foram empregados em colunas cromatogrãficas comerciais, contendo sílica, C18, C8 e MIP (polímeros de impressão molecular) (EP2156182; O2008148547; US20040191537 ; US6284250; WO2002101362 ; EP1399726; EP1053021 WO1999039739 PI1020120153394) . Nartotubos de carbono restritos â ligação com macromoléculas por meio de recobrimento com albumina {RACNT-BSA) Restricted access materials (RAM) are newly proposed, silica-based sorbents, and provided with pores where the various low molecular weight molecules are capable of binding. In addition, such materials have their outer surface modified with hydrophilic binders to the point of chemically making the binding of undesired macro-molecules present in the matrix chemically unviable (Sadılek P., Satinsky D., Solich P., Anal. Chem., 26 (2007) 375- 384.). RAMs have been used in the direct extraction of organic analytes in protein fluids such as blood, plasma, serum, etc. Examples of applications of restricted access materials in analytical methods have been observed for the extraction of organic compounds using chromatographic columns and pre-column chromatography. As a solid reagent, RAMs were already employed in commercial chromatography columns containing silica, C18, C8 and MIP (molecular printing polymers) (EP2156182; O2008148547; US20040191537; US200281050; WO2002101362; EP135721; EP1053021; WO1999039739; PI1020120153394). Carbon atoms restricted to macromolecule binding by coating with albumin (RACNT-BSA)
[005] Como pôde ser visto, os CNT são materiais de alta capacidade adsortiva, pouco seletívos e que apresentam uma capacidade indesejada de reter macromoléculas, inviabilizando sua aplicação direta na extração de analitos em fluidos proteicos. Contudo, cabe ressaltar que não hã na literatura nenhum relato de CNT capazes de eliminar macromoléculas, sendo esses materiais promissores na separação/extração/pré-concentração direta de analitos orgânicos e inorgânicos em fluidos proteicos .  As can be seen, CNTs are highly adsorptive materials, which are not very selective and have an undesirable ability to retain macromolecules, making their direct application in the extraction of analytes in protein fluids impossible. However, it should be noted that there are no reports in the literature of CNT capable of eliminating macromolecules, and these materials are promising in the direct separation / extraction / preconcentration of organic and inorganic analytes into protein fluids.
[006] O objetivo principal da invenção é descrever o processo de obtenção e o uso de um CNT restrito à ligação de macromoléculas (RACNT-BSA) . Para isso, CNTs foram recobertos/encapsulados por uma camada de albumina bovina sérica quimicamente entrecruzada. A extração direta de cádmio em soro sanguíneo humano seguido de análise por absorção atómica é descrita como modelo nessa patente. Contudo, quaisquer outros analitos inorgânicos ou orgânicos podem ser extraídos de fluídos proteicos empregando os RACNT-BSA. Até o momento, não há relatos em bancos de dados sobre a tecnologia desenvolvida. Os RACNT-BSA tem potencial para ampla utilização e comercialização em indústrias e laboratórios que trabalham com análises de metais e compostos orgânicos em fluídos biológicos, podendo ser acondicionados em qualquer sistema de extração que utilize sorventes sólidos, com a grande vantagem de ser possível a injeção direta de fluidos proteicos sem qualquer etapa prévia de desproteinização. Objeto da Invenção: The main object of the invention is to describe the method of obtaining and using a CNT restricted to macromolecule binding (RACNT-BSA). For this, CNTs were coated / encapsulated by a layer of chemically cross-linked serum bovine albumin. Direct extraction of cadmium into human blood serum followed by atomic absorption analysis is described as a model in that patent. However, any other inorganic or organic analytes can be extracted from protein fluids using the RACNT-BSA. To date, there are no reports in databases on the technology developed. RACNT-BSA has the potential for wide use and commercialization in industries and laboratories that work with analyzes of metals and organic compounds in biological fluids, and can be packaged in any extraction system that uses solid sorbents, with the great advantage of being possible the injection directly from protein fluids without any previous deproteinization step. Object of the Invention:
[007] O objetivo da presente invenção consiste no desenvolvimento e o uso de nanotubos de carbono restrito à ligação com macromoléculas por meio de recobrimento com albumina (RACNT-BSA) , e que sejam capazes extrair diretamente analitos presentes em matrizes proteicas sem a necessidade prévia de decomposição da matriz por procedimentos morosos como decomposição por micro-ondas, via seca, via úmida, etc. Esses materiais são capazes de eliminar cerca de 100% das macromoléculas presentes em fluidos protéicos submetidos à extração. O princípio de eliminação das macromoléculas provém de natureza físico- química do sorbente, uma vez que a imobilização da BSA na superfície dos CNT bloqueia os sítios de ligação do CNT impedindo ligações do mesmo com as proteínas da amostra. Além disso, a extração de amostras com pH diferente do ponto isoelêtrico da camada externa de BSA e das proteínas da amostra, faz com que ambas as proteínas (da amostra e do revestimento do CNT) estejam ionizadas positivamente (pH abaixo do ponto isoelético) ou negativamente (pH acima do ponto isoelêtrico) , resultando em repulsão eletrostãtica, e impedindo que as proteínas da amostras sejam aderidas à capa de BSA que envolve o CNT. O RACNT-BSA apresenta longo tempo de vida útil, comprovado pela manutenção do seu desempenho mesmo após cerca de 300 extrações consecutivas de amostras de soro sanguíneo humano para análise de cádmio. Em termos de custo, para a produção de 1 g de RACNT-BSA são requeridos cerca de R$ 150,00. Vale ressaltar que no RACNT-BSA proposto nesse pedido de proteção por patente não se assemelha as patentes relatadas sobre CTN. Breve Descrição das Figuras The aim of the present invention is the development and use of carbon nanotubes restricted to binding to macromolecules by coating with albumin (RACNT-BSA), and which are capable of directly extracting analytes present in protein matrices without the prior need of decomposition of the matrix by slow procedures such as microwave decomposition, dry route, humid route, etc. These materials are capable of eliminating about 100% of the macromolecules present in protein fluids undergoing extraction. The principle of elimination of the macromolecules comes from the physico-chemical nature of the sorbent, since the immobilization of the BSA on the surface of the CNT blocks the CNT binding sites preventing binding of the same to the proteins of the sample. In addition, extraction of samples with a pH different from the isoelectric point of the outer layer of BSA and of the sample proteins, causes both the proteins (from the sample and from the CNT coating) to be positively ionized (pH below the isoelectric point) or negatively (pH above the isoelectric point), resulting in electrostatic repulsion, and preventing the sample proteins from being adhered to the BSA envelope surrounding the CNT. RACNT-BSA has a long shelf-life, proven by maintaining its performance even after about 300 consecutive extractions of human serum samples for cadmium analysis. In terms of cost, the production of 1 g of RACNT-BSA requires about R $ 150.00. It is noteworthy that in the RACNT-BSA proposed in this application for patent protection does not resemble the patents reported on CTN. Brief Description of the Figures
[008] Figura 1: Esquema representativo da síntese do RACNT-BSA. (1) CNT, (2) reagentes responsáveis pela funcionalização, {3) reação de funcionalização, (4) CNT funcionalizados, (5) BSA (albumina de soro bovino), (6) etapa de recobrimento dos CNT, (7 e 8) RACNT-BSA.  [008] Figure 1: Representative scheme of RACNT-BSA synthesis. (1) CNT, (2) reagents responsible for functionalization, (3) functionalisation reaction, (4) functionalized CNTs, (5) BSA (bovine serum albumin), (6) CNT coating step, (7 and 8 ) RACNT-BSA.
Descrição Detalhada da Invenção  Detailed Description of the Invention
[009] Na síntese de RACNT-BSA, os CNT de paredes simples ou múltiplas podem ser recobertos diretamente ou após uma reação de funcionalização. Os CNT não modificados apresentam características apoiares e são empregados principalmente para extração/pré-concentração de compostos orgânicos. Para analitos inorgânicos e moléculas mais polares, a funcionalização do material proporciona melhores características. Nesta etapa, os CNT podem ser funcionalízados com diferentes moléculas orgânicas usando estratégias de ligação covalente ou não covalente (Antonio V. Herrera-Herrera, et al . Analytica Chimica Acta. 734 (2012) 1) . As principais funcionalizações são:  In the synthesis of RACNT-BSA, single or multiple-walled CNTs can be coated either directly or after a functionalization reaction. Unmodified CNTs exhibit supportive characteristics and are mainly used for extraction / preconcentration of organic compounds. For inorganic analytes and more polar molecules, the functionalization of the material provides better characteristics. In this step, the CNTs can be functionalized with different organic molecules using covalent or non-covalent binding strategies (Antonio V. Herrera-Herrera et al., Analytica Chimica Acta, 734 (2012) 1). The main functionalisations are:
[0010] Oxidação: uso de reagentes oxidantes para formação de grupamentos -OH, -C=0 e -COOH. Os reagentes empregados são: NH3. H20/H202 ; H2S04/ H202; HN03; KMN04; 2Cr207; HN03/ ¾02. Oxidation: Use of oxidizing reagents for formation of -OH, -C = O and -COOH groups. The reagents used are: NH 3 . H 2 O / H 2 O 2 ; H 2 SO 4 / H 2 0 2 ; HNO 3 ; KMNO 4 ; 2CH 2 0 7; HNO 3 / ¾0 2 .
[0011] Outras modificações de CNT oxidados: Esta funcionalização envolve inicialmente a oxidação dos CNTs e posteriormente a reação com aminas, tais como: etilenodiamina (EDA) (Z. Zang, Z. Hu, Z. Li, Q. He, X. Chang, J. Hazard. Mater. 172 (2009) 958); Trís(2» aminoetil) amina (TAA) (Y. Cui, S. Liu, Z.-J. Hu, X.-H. Liu, H.~W. Gao, Microchim. Acta 174 (2011) 107); 2- aminobenzotiazole {ABT ) (R. Li, X. C ang, Z. Li, 2. Zang, Z. Hu, D . Li, Z. Tu, icrochim. Actal72 (2011) 269); ácido iminodiacético (IDA) (J. Wang, X. Ma, G. Pang, M. Pan, X. Ye, S. Wang, J. Hazard. Mater. 186 (2011) 1985) . Outros tipos de funcionalizaçâo envolvem a criação de ligação dos grupamentos amida com aminoácidos como, L-cisteína (Y. Liu, Y. Li, X.-P. Yan, Adv. Funct . Mater. 18 (10) 1536), L- tirosina (P.H. Pacheco, P. Smichowski, G. Polia, L.D. Martinez, Talanta 79(2009) 249) e L-alanina (M. Savio, B. Parodi, L.D. Martinez, P. Smichowski, R.A. Gil, alanta 85 (2011) 245) . Other modifications of oxidized CNTs: This functionalization initially involves the oxidation of CNTs and subsequent reaction with amines, such as: ethylenediamine (EDA) (Z. Zang, Z. Hu, Z. Li, Q. He, X. Chang, J. Hazard, Mater, 172 (2009) 958); Tris (2 ' aminoethyl) amine (TAA) (Y. Cui, S. Liu, Z.-J. Hu, X.-H. Liu, H. W .; Gao, Microchim. Acta 174 (2011) 107); 2- aminobenzothiazole (ABT) (R. Li, X. Ang., Z. Li. 2. Zang. Z. Hu. D. Li. Z. Tu.chrochim.Atal72 (2011) 269); iminodiacetic acid (IDA) (J. Wang, X. Ma, G. Pang, M. Pan, X. Ye, S. Wang, J. Hazard, Mater. 186 (2011) 1985). Other types of functionalization involve the creation of amide group linkages with amino acids such as L-cysteine (Y. Liu, Y. Li, X.-P. Yan, Adv. Funct. Mater. 18 (10) 1536), L- tyrosine (PH Pacheco, P. Smichowski, G. Polia, LD Martinez, Talanta 79 (2009) 249) and L-alanine (M. Savio, B. Parodi, LD Martinez, P. Smichowski, RA Gil, ) 245).
[0012] Outra forma de funcionalizar os CNT pode ser obtida pelo uso de plasma induzido de N2. Esta técnica permite a formação de grupos ati os de - C* e - C-N* nos CNT que posteriormente podem reagir com outros compostos orgânicos e receberem outros grupos funcionais. Esta técnica visa substituir a etapa prévia de oxidação, usada em outras funcionalizações dos CNT, por apresentar maior eficiência jã que a etapa de oxidação podem degradar os CNT. As principais modificações com esta técnica envolvem o uso de poliacrilamida (PAAM) , poli (N,N~dimetilacrilamida) PDMA (D. Shao, X. Ren, J. Hu, Y. Chen, x. Wang, Colloids Surf., A 360 (2010) 74); vinilpiridina (VP) (X. Ren, D. Shao, G. Zhao, G. Sheng, J. Hu, S. Yang, X. Wang, Plasma Process. Polym. 8 (2011) 589) e polissacarídeos (CMC) (D. Shao, Z. Jiang, X. Wang, J. Li, Y. Meng, J. Phys. Chern. BAnother way of functionalizing CNTs can be obtained by the use of N 2 induced plasma. This technique allows the formation of α - C * and - CN * groups in CNTs which can then react with other organic compounds and receive other functional groups. This technique aims to replace the previous stage of oxidation, used in other functionalisations of the CNT, to present higher efficiency since the oxidation stage can degrade the CNT. The main modifications with this technique involve the use of polyacrylamide (PAAM), poly (N, N-dimethylacrylamide) PDMA (D. Shao, X. Ren, J. Hu, Y. Chen, X. Wang, Colloids Surf., A 360 (2010) 74); (VP) (X. Ren, D. Shao, G. Zhao, G. Sheng, J. Hu, S. Yang, X. Wang, Plasma Process Polym. 8 (2011) 589) and polysaccharides (CMC) ( D. Shao, Z. Jiang, X. Wang, J. Li, Y. Meng, J. Phys. Chern.
113 (2009) 860) . 113 (2009) 860).
[0013] A adição radicalar também possibilita a funcionalização com ácido fenil-iminoacético (PIDA) (J.L. Bahr, J.M. Tour, Chem. Mater. 13 (2001) 3823) . [0014] Nessa patente descrevemos a síntese de um RACNT- BSA avaliado como sorvente na extração direta de cádmio em soro sanguíneo humano. Os CNT de paredes simples ou múltiplas e ácido nítrico foram usados como reagente sólido e agente oxidante, respectivamente. As quantidades empregadas foram de 0,005 a 100 g de nanotubos de carbono e de 0,01 a 300 mL de ácido nítrico. Os reagentes foram misturados em um balão de destilação. Posteriormente, foi montado um sistema de refluxo e o balão foi aquecido em manta de aquecimento de 40 e 180 °C, entre 20 e 200 minutos. Em seguida, o material foi filtrado e lavado com água e etanol até que o filtrado apresentasse pH superior a 4,00 e inferior a 9,00: Radical addition also enables functionalization with phenylimininoacetic acid (PIDA) (JL Bahr, JM Tour, Chem. Mater. 13 (2001) 3823). In this patent we describe the synthesis of a RACNT-BSA evaluated as a sorbent in the direct extraction of cadmium in human blood serum. Single or multiple-walled CNTs and nitric acid were used as solid reagent and oxidizing agent, respectively. The amounts employed were 0.005 to 100 g of carbon nanotubes and 0.01 to 300 mL of nitric acid. The reactants were mixed in a distillation flask. Subsequently, a reflux system was set up and the flask was heated in a heating mantle of 40 and 180øC, between 20 and 200 minutes. The material was then filtered and washed with water and ethanol until the filtrate had a pH greater than 4.00 and less than 9.00:
[0015] Em seguida, os CTN funcionalizados foram recobertos com BSA (Menezes M. L . , Félix G, , J. Liq. Chromatogr R. T. , 21 (1998) 2863; de Lima V. V., Cassiano N. M., Cass Q. B . , Química Nova, 29 (2006) 72), sendo importante enfatizar que qualquer outro tipo de CNT funcionalízado por ser submetido a esse processo de recobrimento. Percolou-se então, sequencialmente, entre 10 e 50 mL de solução de 0,1 e 10% (m/v) de BSA em tampão fosfato 0,01 a 0,5 mol L"1 (pH de 5 a 9) e de l a 10 mL de uma solução aquosa de 5 a 50 % (v/v) glutaraldeído em um cartucho contendo de 20 a 1500 mg do CTN. Nessa etapa, o gluteraldeído, que é um agente reticulante bifuncional, estabeleceu interligações entre os grupamentos amina das cadeias proteicas, fixando a camada de BSA ao redor do CNT. Transcorrido um período de 1 a 20 h, um volume de 1 a 50 mL de uma solução aquosa de boroidreto de sódio de 0,05 a 5% (m/v) foram percolados pelo cartucho com a finalidade de reduzir as iminas (originadas da reação anterior) a aminas que são grupamentos mais estáveis quimicamente,, conferindo assim estabilidade química ao material . Finalmente o RACNT- BSA foi lavado em abundância com agua par a retirada dos reagentes remanescentes do processo de recobrimento . The functionalized CTNs were then coated with BSA (Menezes M.L., Felix G, J. Liq. Chromatogr RT, 21 (1998) 2863; Lima VV, Cassiano NM, Cass Q.B., Química Nova, 29 (2006) 72), and it is important to emphasize that any other type of CNT functioned for being subjected to this coating process. Then, between 10 and 50 ml of a 0.1 and 10% (w / v) solution of BSA in phosphate buffer 0.01 to 0.5 mol L -1 (pH of 5 to 9) and of 10 mL of an aqueous solution of 5 to 50% (v / v) glutaraldehyde in a cartridge containing 20 to 1500 mg of the CTN. In this step, gluteraldehyde, which is a bifunctional crosslinking agent, established interconnections between the amine of the protein chains, securing the BSA layer around the CNT. After a period of 1 to 20 h, a volume of 1 to 50 mL of an aqueous solution of sodium borohydride of 0.05 to 5% (w / v) were percolated by the cartridge for the purpose of reducing the imines (originating from the previous reaction) to amines which are more chemically stable moieties, thereby conferring chemical stability on the material. Finally the RACNT-BSA was washed in abundance with water to remove the remaining reagents from the coating process.
[0016] Terminado o recobrimento, uma coluna de 2 a 7 mm de diâmetro interno po l a 5 cm de comprimento foi preenchida com o RACNT-BSA e acoplada em sistema cromatogrãfico na posição da coluna analítica. 25 pL de um padrão aquoso de BSA 44 mg.mL'1 (mesma concentração proteica do soro sanguíneo humano) foram injetados pela coluna e o pico resultante (detectado a 254 nm) foi comparado com o pico obtido da injeção do mesmo padrão de BSA no sistema sem a coluna. Verificou- se então que praticamente não houve retenção de albumina no RACNT-BSA (com recuperação de 101 ± 1%) , indicando que o recobrimento impediu, satisfatoriamente, a ligação de proteínas na superfície do CNT. Vale ressaltar que o mesmo experimento foi também executado com o CNT não recobertos, resultando em uma retenção de cerca de 99 ± 1 % das proteínas injetadas (recuperações menores que 1 %) , e comprovando a importância da camada fixa de BSA na eliminação de proteínas . After the coating was complete, a column of 2 to 7 mm internal diameter by 5 cm in length was filled with RACNT-BSA and coupled in a chromatographic system to the analytical column. 25 μl of an aqueous BSA standard 44 mg.mL- 1 (the same human blood serum protein concentration) were injected through the column and the resulting peak (detected at 254 nm) was compared to the peak obtained from the injection of the same BSA standard on the system without the column. It was verified that there was practically no albumin retention in the RACNT-BSA (with recovery of 101 ± 1%), indicating that the coating satisfactorily prevented the binding of proteins on the CNT surface. It is noteworthy that the same experiment was also performed with the CNT uncoated, resulting in a retention of about 99 ± 1% of the injected proteins (recoveries less than 1%), and confirming the importance of the fixed layer of BSA in the elimination of proteins .
[0017] A coluna contendo o RACNT-BSA foi então acoplada na posição da alça de amostragem em um injetor comutador manual empregado no sistema de pré-concentração em fluxo, com eluição d reta para um equipamento de absorção atómica com tubo de níquel aquecido (TS -FF-AAS, do inglês thermospray flame furnace atomic absorption spectrometry) . O sistema foi otimizado e validado para extração de cádmio em soro e plasma sanguíneo humano. 0 método mostrou-se eficiente na análise direta (sem a necessidade de qualquer etapa preliminar de preparo de amostras) de cádmio, com limite de quantificação de 0,8 ug/L>. Ademais, o material, além de permitir a extração direta de cádmio, ainda apresentou alto poder de pré-concentração (fator de pré- concentração de 5,7). Por fim, o mesmo cartucho foi empregado durante cerca de 300 extrações consecutivas, não apresentando diferença significativa em sua eficiência de eliminação de macromoléculas, bem como na extração de cádmio. Esse fato torna a tecnologia atraente no sentido de que o custo por análise pode ser drasticamente reduzido já que o mesmo cartucho foi reutilizado várias vezes, além do fato de que o sistema evita possíveis erros analíticos que ocorrem nas etapas de preparo de amostra como digestão da matéria orgânica. Outras características do sistema são: consumo reduzido de reagentes (como ácidos e peróxidos usados nas etapas de digestão das amostras) ; ausência de equipamentos sofisticados para digestão das amostras (digestores de micro-ondas) e a possibilidade de automação da análise em sistemas em fluxo ou cromatográficos . The RACNT-BSA-containing column was then coupled at the position of the sampling loop on a manual switch injector employed in the flow preconcentration system, eluted straight to a heated nickel tube atomic absorption equipment ( TS-FF-AAS, from thermospray flame furnace atomic absorption spectrometry). The system was optimized and validated for cadmium extraction in serum and human blood plasma. The method was efficient in the direct analysis (without the need of any preliminary step of sample preparation) of cadmium, with limit of quantification of 0.8 ug / L>. In addition, the material, besides allowing the direct extraction of cadmium, still presented high preconcentration power (preconcentration factor of 5.7). Finally, the same cartridge was used during about 300 consecutive extractions, presenting no significant difference in its efficiency of elimination of macromolecules, as well as in the extraction of cadmium. This fact makes the technology attractive in that the cost per analysis can be drastically reduced since the same cartridge has been reused several times, in addition to the fact that the system avoids possible analytical errors that occur in the steps of sample preparation as digestion of the organic matter. Other characteristics of the system are: reduced consumption of reagents (such as acids and peroxides used in the digestion of samples); absence of sophisticated equipment for digestion of samples (microwave digesters) and the possibility of automation of the analysis in flow or chromatographic systems.

Claims

REIVINDICAÇÕES
1. Nanotubos de carbono restritos à ligação com macromoléculas por meio de recobrimento com albumina 1. Carbon nanotubes restricted to binding to macromolecules by coating with albumin
- (RACNT-BSA) , caracterizado por utilizar metodologia de síntese do material . - (RACNT-BSA), characterized by the use of material synthesis methodology.
2. Nanotubos de carbono restritos à ligação com macromoléculas por meio de recobrimento com albumina (RACNT-BSA) , caracterizado por ser utilizado em extração de fase sólida e recheio de pré-colunas e colunas cromatogrãfica.  2. Carbon nanotubes restricted to binding to macromolecules by means of albumin coating (RACNT-BSA), characterized in that it is used in solid phase extraction and filling of precolumns and chromatographic columns.
3. Nanotubos de carbono restritos à ligação com macromoléculas por meio de recobrimento com albumina (RACNT-BSA) , de acordo com a reivindicação 1, caracterizado por utilizar nanotubos de carbono de paredes simples e múltiplas.  3. Carbon nanotubes restricted to macromolecule binding by albumin coating (RACNT-BSA) according to claim 1, characterized in that the use of single and multiple-walled carbon nanotubes.
4. Nanotubos de carbono restritos à ligação com macromoléculas por meio de recobrimento com albumina (RACNT-BSA) , de acordo com a reivindicação 1, caracterizado por utilizar agentes oxidantes dos seguintes grupos: NH3 , H20/H202 ; H2S04/ H202 ; HN03 ; KMN04 ; Cr207-2; HN03/ H202; Cr04-2.  Carbon nanotubes restricted to macromolecule binding by albumin coating (RACNT-BSA) according to claim 1, characterized by using oxidizing agents of the following groups: NH 3, H 2 O / H 2 O 2; H2SO4 / H2O2; HNO3; KMN04; Cr207-2; HNO3 / H2O2; Cr04-2.
5. Nanotubos de carbono restritos à ligação com macromoléculas por meio de recobrimento com albumina (RACNT-BSA) , de acordo com a reivindicação 1, caracterizado por utilizar reagentes de funcionalização dos seguintes grupos: Tris (2-aminoetil) amina (TAA) ; 2-aminobenzotiazole (ABT ) ; ácido iminodiacético (IDA) L~cisteína; L~tirosina; L-alanína; políacrílamída (PAAM) ; poli(N,N- dimetilacrilamida) PDMA vinilpiridina (VP) polissacarídeos (CMC) ; fenil-iminoacético (PIDA) . Carbon nanotubes restricted to macromolecule binding by albumin coating (RACNT-BSA) according to claim 1, characterized in that use of functionalizing reagents of the following groups: Tris (2-aminoethyl) amine (TAA); 2-aminobenzothiazole (ABT); iminodiacetic acid (IDA) L-cysteine; L-tyrosine; L-alanine; polyacrylamide (PAAM); poly (N, N-dimethylacrylamide) PDMA vinylpyridine (VP) polysaccharides (CMC); phenyl-iminoacetic acid (PIDA).
6. Nanotubos de carbono restritos à ligação com macromoléculas por meio de recobr mento com albimina (RACNT-BSA) , de acordo com a reivindicação 1, caracterizado por utilizar uma das seguintes proteínas: albumina, glicoproteínas, proteínas do leite. Carbon nanotubes restricted to macromolecule binding by albumin recovery (RACNT-BSA) according to claim 1, characterized in that one of the following proteins is used: albumin, glycoproteins, milk proteins.
7. Nanotubos de carbono restritos à ligação com macromoléculas por meio de recobrimento com albumina (RACNT-BSA) , de acordo com a reivindicação 1, caracterizado por utilizar reagentes oxidantes na faixa de concentração de 2 a 100%.  Carbon nanotubes restricted to macromolecule binding by albumin coating (RACNT-BSA) according to claim 1, characterized in that the use of oxidizing reagents in the concentration range of 2 to 100%.
8. Nanotubos de carbono restritos â ligação com macromoléculas por meio de recobrimento com albumina (RACNT-BSA) , de acordo com a reivindicação 1, caracterizado por utilizar reagentes de funcíonalização na faixa de concentrações de 0,0001 a 5 mol.L"1. 8. Carbon nanotubes restricted to macromolecule binding by albumin coating (RACNT-BSA) according to claim 1, characterized in that the use of functional reagents in the concentration range of 0.0001 to 5 mol L -1 .
9. Nanotubos de carbono restritos à ligação com macromoléculas por meio de recobrimento com albumina (RACNT-BSA) , de acordo com a reivindicação 1, caracterizado por utilizar BSA na faixa de concentrações de 0,1 a 50 % (m/v) .  9. Carbon nanotubes restricted to macromolecule binding by albumin coating (RACNT-BSA) according to claim 1, characterized by using BSA in the concentration range of 0.1 to 50% (m / v).
10. Nanotubos de carbono restritos à ligação com macromoléculas por meio de recobrimento com albumina (RACNT-BSA) caracterizado por servir como extrator em fase sólida de composto orgânico e inorgânico e como recheio de colunas e pré-colunas cromatograficas .  10. Carbon nanotubes restricted to macromolecule binding by coating with albumin (RACNT-BSA) characterized by serving as a solid phase extractor of organic and inorganic compound and as a filling of columns and chromatographic pre-columns.
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