Course: Nanotechnology and Nanosensors

By Prof. Hossam Haick, Ph.D.

Edilson Gomes de Lima

HOW PRODUCE ARTIFICIAL OR SYNTHETIC ANTIBODY FOR NANOSENSOR – WITH EXAMPLE USES FOR HUMAN SENSE

A preliminary study based in nanotechnology procedures

Presentation

Edilson Gomes de Lima – Mechanical engineer and chemical

Edoardo Zunino – Chemical engineer and master’s degree student

Silvia Demuru – Student in biomedical engineer

Edoardo Zunino. Sex: Male. Date of birth 08/05/1991. Nationality Italian. From March 2014 Student in chemical engineering master’s degree at Polytechnic of Milan. From March to June 2015 Assistant WAME&Expo2015, Expo2015, Rho Milan, Italy. From September to February 2014 Apprenticeship at Iplom s.p.a. for bachelor’s thesis on hydrocarbon reactor optimization. Form 2012 to 2015 work as cook in a restaurant. From September 2010 to February 2015 Student in chemical engineering bachelor’s degree at Polytechnic School of Genoa. 2010 graduation at Liceo Scientifico Blaise Pascal (secondary school), Ovada (Al), Italy Driving licence B, generic skills in economy and finance. Language skills: English, German, Russian and Italian.

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Israel / USA

2015

Nanotechnology and nanosensors

Final Work

HOW PRODUCE ARTIFICIAL OR SYNTHETIC ANTIBODY FOR NANOSENSOR – WITH EXAMPLE USES

CONTENTS

1..ABSTRACT……………………………………...................................................................02

2. DEFINITION………………………………………………..…………………...................03

3. FABRICATION PROCESS..................................................................................................03

4. USES AS NANOSENSOR…………………………………. ………………….................03

5. ANTIBODY……………………………………...………………….…..............................04

6. ANTIBODY SYNTHESIS...................................................................................................05

7. GRAPHENE PRODUCTION………..………....................................................................05

8. GRAPHENE FILM TRANSFER……………………………………………………….....07

9. GRAPHENE FUNCTIONALIZATION…………………………………………………..07

10. AUTONOMATION....…………………………………………………………………...08

11. THE HARDWARE………………………………………………………………………09

12. SKINCADHESION……………………………………………………………………...10

13. CONCLUSION...................................................................................................................10

14. REFERENCES………………….…………………………...............................................10

1. ABSTRACT

This paper presents the process how to produce artificial or synthetic antibody for nanosensor and shows an example for use it as a nanosensor for improvement a human

sense, in the case, the monitoring disease skin and show by electronic ways the results. A useful for this sense is monitoring skin in person without movements or in hospitals. Beyond this paper try to shows

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that the good understand of how to produces, artificial or synthetic antibody or same synthetize natural antibody for nanosensor uses is perfectly adaptable for several medicinal uses. In addition, it is highly adjustable for several nanosensors as a way to improve or adds new senses in humans. This paper have the purpose to be a simple template of how make antibodies for human senses.

2. DEFINITIONS

Here will be presenting a fast and concise presentation about artificial or synthetic antibodies to our uses for nanosensors for adding new human senses.

Which is the simpler or better definition for artificial antibody? An artificial antibody has the goal to imitate natural antibodies, general it bond on a target cell and act on it, or improve natural antibodies for the same purpose. It can also act to recognise a specific molecule. They are mainly use against HIV or Cancer.

Which the great details about antibody know or about in a minimum background or readings about it? In simple words, have learned structure, some function as biosensors and diagnostic, and now, complementary with studies of how they can be use in therapy.

Which kind of antibody you know? They can be classified for structure: IgE, IgD, IgM, IgG, IgA.

Which synthesis more known for makes artificial or syntactic antibodies? If we refer to only bio-particle the most famous is molecular imprinting. However, in case we would to use metal nanoparticles aerosol process & reactor, or if you want to use graphene CVD reactor. In addition, if you want "a full natural" industrial process for antibody production I am actually studying industrial microbiology, which consist to use cells like reactors.

Some example or know about some functionalization for antibody? For the nomenclature here we refer to World Health Organisation’s International Non-

proprietary Names and the United States Adopted Names.

Would be know the most technical names around artificial antibody? As here mentioned in point two, it is possible to use carbon's nanotube produced by CVD process or spherical nanoparticles from aerosol reactor and functionalizing it whit antibodies. On the other hand, synthetic antibodies are protein polymers and it is possible to manage it in order to obtain the desired effect. (I have a lot of material for functionalization of polymers in pharmaceutical field if it is necessary but it is not focus on antibodies in specific).

The antibodies are synthetized with biological material of being lives or with chemicals, in fluid or solid methods? They are produced by cells. We have also solid and particle, so the best way to describes process is to consider a no-Newtonian fluid.

Afters synthetized the particles can be "glued" “Bonded” by chemical affinity in some thin paper or place? The antibodies are specific for a target molecule do to their weak electrostatic interaction. For this reason they show affinity only to a precise molecule.

3. FABRICATION PROCESS

Will be used CVD process as synthesis of graphene that will be our base for the antibodies. So as production we will uses four process: CVD, fluidic synthesis, and Langmuir-Blodgget process, and too functionalization process and bond the antibody by bond chemical process. Like scheme previously showed in image 1:

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Image 1 – Like the process of microarray where could be used nanoparticles functionalized like nanorrods.

4. USES AS NANOSENSOR

The nanosensor for our project will be a sticker to monitoring skin. For a good interaction of communication between atoms and human we need uses electrical signals readers for our reach. So we need use the flash memory as hardware, and software with previously data base with analysts needed.

5. ANTIBODY

Antibody structure and function.

The function of an antibody is to bind foreign or non-self-molecules. The host can produce a large array of antibodies, structurally similar (all are Y form) yet unique. Antibodies come in millions of different amino acid sequences and are the most diverse proteins known. The chemical structure of antibodies explain three different functions:

1) Binding versatility 2) Binding specificity 3) Biological activity

The basic structure of an immunoglobulin is outlined in Fig.1. The antibody molecule has four polypeptide chains, two heavy (H) chains with molecular weights of 50kDa and two light (L) chains (25kDa) linked by disulphide bonds.

Fig. 1. Antibody structure: typical IgG molecule structure. Antigen binding sites are indicated by the yellow triangles. Red lines indicate disulphide bridge.

The chains have both constant (C) and variable (V) regions. The H chain has one variable region (VH) that is responsible for antigen binding and three constant regions (CH1, CH2 and CH3). The light chain has one variable region (VL), which is an important part of the antigen- binding site, and one constant region (CL).

There are five classes of immunoglobulin which are distinguished by their heavy chains: IgA, IgG, IgM, IgD and IgE. Class switching of the heavy chain during gene rearrangement gives

rise to the isotype of the immunoglobulin. There are two types of light chains, kappa and lambda, which combine with heavy chains to form a complete antibody molecule. IgG is the predominant class of antibody produced during the matured immune response and is the most widely targeted for immune library construction [8]. The Fc region consists of three domains, CH1, CH2 and CH3 that confer effector functions, such as complement activation, on the antibody. Antigen binding is mediated by the variable light (VL) and heavy (VH) domains which bring together the hyper-variable regions of the antibody, known as the complementarity determining regions (CDRs). The antibody constant regions are generally conserved with only small differences in sequence being found in the various anti- body classes. However, the CDRs exhibit a high level of sequence diversity. The diversity of antibodies in the immune system is achieved by gene recombination and somatic hyper-mutagenesis of the encoding genes. The encoding gene segments are outlined in Fig.2. In vertebrate genomes, there are 11 Constant (CH), 123–129 Variable (VH), 27 Diverse (DH) and 9 Joining (JH) gene segments that combine to encode the heavy chains. During B-cell development, the immunoglobulin loci undergo rearrangements. Within the heavy chain locus, the VH-DH-JH rearrange and this exon becomes linked to a combination of CH segments during transcription. Subsequently, the mRNA is translated into the immunoglobulin isotype that is specific

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Fig. 1. Biosensor components. (a) Analyte interaction with biorecognition element: this is facilitated by the specificity of the immobilised antibody for its cognate antigen(purple). Other biorecognition elements include enzymes, lectins, receptors and microbial cells. (b) Signal transduction: converts the interaction of the analyte molecule andanalyte into a quantifiable signal. (c) Readout or display: shows the specific signal generated by interaction with the analyte of interest. Yellow: non specific analyte.

Fig. 2. Typical IgG molecule structure. Antibody molecule (∼150 kDa). The antigenbinding sites are indicated by the triangles and the disulphide bridges are indicatedby red lines.

antibody-based recognition elements, have been developed on awide range of transduction platforms for a multitude of analytes.The transducer element translates the selective recognition of theanalyte into a quantifiable signal and thus, has major influence onsensitivity [15]. Transduction approaches include electrochemical,piezoelectric and optical systems [16].

2.2. Transduction platforms

This section briefly introduces some of the transduction plat-forms commonly exploited in biosensors incorporating antibody-based biorecognition. Monoclonal and polyclonal antibodies havebeen successfully employed and are the predominant antibodyform used in biosensors. However, as discussed later, the impor-tance of recombinant antibodies for biosensor applications isgaining increasing significance. Table 1 lists several examples oftransduction elements, utilising both polyclonal and monoclonalantibodies for the detection of various analytes. The references alsoprovide additional detailed information on the principles underpin-ning each of the transduction methods and are comprehensivelyreviewed by Jiang and co-workers [15] with respect to pesticidesand both Luppa and co-workers [17] and D’Orazio [13] with respectto clinical uses.

Fig. 3. Genes encoding human antibodies. Immunoglobulin loci: Heavy chain (chromosome 14), ! light chain (chromosome 2) and " light chain (chromosome 22). Variable(V), Constant (C), Joining (J) and Diverse (D) segment genes are shown. In immature B cells V-D-J segments are rearranged and linked to C# to produce mRNA encoding forIgM or C$ to give mRNA encoding for IgD. mRNA encoding IgG is produced by recombination resulting in the bringing together of C%2, C%4 C& and C'2 by the deletion of thefive CH segments. This figure was adapted from [8,10].

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to the lymphocyte. A similar chain of events occurs for the kappa and lambda loci, with the absence of D segments. The diversity introduced into the immune system by these rearrangements is increased further by somatic hypermutation (SHM). SHM introduces errors into the genes encoding the variable regions of individual B-cells. During the narrow time frame in the proliferation of B-cells, the locus undergoes an extremely high rate of mutation, predominantly base substitution, approximately 1 million times higher than the spontaneous rate of mutation across the genome. The introduction of random mutations gives rise to antibodies that lose their affinity for the antigen and undergo cell death, or generates those advantageous antibodies, where there is an affinity increase and subsequent proliferation of the associated antibody-producing clone of lymphocytes.

Fig. 2. Genes encoding human antibodies.

6. ANTIBODY SYNTHESIS

It was the pioneering work of G. Kohler and C. Mil- steinf5’ that first made the in-vitro synthesis of a mono- clonal antibody with the desired specificity and in almost any amount possible. They obtained B-lymphocytes from the spleen of mice specifically immunized against sheep erythrocytes, a cellular antigen, and then brought them into close contact with a mouse myeloma cell line (X63- Ag8) along with a fusion-promoting agent (Sendai virus; polyethylene glycol 4000; dimethylsulfoxide), thus achieving cell fusion (hybridization). The fusion mixture was cultivated in a selective culture medium (HAT medium, containing hypoxanthine, aminopterine, and thymidine), in which only hybridoma cells are able to grow. Although the myeloma tumor cell is immortal and grows in a culture medium, it does not have the enzyme hypoxanthineguanine

phosphoribosyltransferase (HGPRT) and cannot survive in a HAT medium. Only those cells can multiply in a HAT medium which contain the enzyme HGPRT, which enables exogenously supplied hypoxanthine to be used for the salvage pathway of purine nucleotide synthesis. Aminopterine, an antagonist of folic acid reductase, blocks the main synthesis pathway for thymidine. Hybridoma cells are HGPRT+ and also contain the enzyme thymidine kinase (they are TIC+): thus, they can make use of exogenously supplied hypoxanthine and thymidine. This is also of significance for myeloma cell line variants selected on account of a thymine kinase defect. The hybrid cells are not all useful producer of monoclonal antibodies at first, since they contain chromosomes of both parental lines and can, in principle, produce two types of both heavy and light antibody chains. However, monoclonal antibody consists of one type of heavy chain, which should only be coded by spleen cell chromosomes, for antibody specificity is selected via the specificity of the fused spleen cells. Hybrid cells lose chromosomes during the first cell division; the number of chromosomes is reduced from about 112 to a more stable number of 70. Thus the proteins that are coded on the lost chromosomes cannot be synthesized. A purging process therefore takes place at this stage of cloning. The useful clones must be especially carefully selected, so that the desired hybridomas and monoclonal antibodies are obtained. Finally, the selected and stabilized clones are continuously multiplied in tissue culture. In order to maintain the stock, parts are frozen in liquid nitrogen; they can thus be store for a practically unlimited period of time and reintroduced into the cell culture at will.

7. GRAPHENE - Graphene production

Graphene can be prepared using mechanical exfoliation, epitaxial growth on SiC, and chemical exfoliation, chemical vapour deposition (CVD) has emerged as an important method for its preparation and production. During CVD process, gas

12 P.J. Conroy et al. / Seminars in Cell & Developmental Biology 20 (2009) 10–26

Fig. 1. Biosensor components. (a) Analyte interaction with biorecognition element: this is facilitated by the specificity of the immobilised antibody for its cognate antigen(purple). Other biorecognition elements include enzymes, lectins, receptors and microbial cells. (b) Signal transduction: converts the interaction of the analyte molecule andanalyte into a quantifiable signal. (c) Readout or display: shows the specific signal generated by interaction with the analyte of interest. Yellow: non specific analyte.

Fig. 2. Typical IgG molecule structure. Antibody molecule (∼150 kDa). The antigenbinding sites are indicated by the triangles and the disulphide bridges are indicatedby red lines.

antibody-based recognition elements, have been developed on awide range of transduction platforms for a multitude of analytes.The transducer element translates the selective recognition of theanalyte into a quantifiable signal and thus, has major influence onsensitivity [15]. Transduction approaches include electrochemical,piezoelectric and optical systems [16].

2.2. Transduction platforms

This section briefly introduces some of the transduction plat-forms commonly exploited in biosensors incorporating antibody-based biorecognition. Monoclonal and polyclonal antibodies havebeen successfully employed and are the predominant antibodyform used in biosensors. However, as discussed later, the impor-tance of recombinant antibodies for biosensor applications isgaining increasing significance. Table 1 lists several examples oftransduction elements, utilising both polyclonal and monoclonalantibodies for the detection of various analytes. The references alsoprovide additional detailed information on the principles underpin-ning each of the transduction methods and are comprehensivelyreviewed by Jiang and co-workers [15] with respect to pesticidesand both Luppa and co-workers [17] and D’Orazio [13] with respectto clinical uses.

Fig. 3. Genes encoding human antibodies. Immunoglobulin loci: Heavy chain (chromosome 14), ! light chain (chromosome 2) and " light chain (chromosome 22). Variable(V), Constant (C), Joining (J) and Diverse (D) segment genes are shown. In immature B cells V-D-J segments are rearranged and linked to C# to produce mRNA encoding forIgM or C$ to give mRNA encoding for IgD. mRNA encoding IgG is produced by recombination resulting in the bringing together of C%2, C%4 C& and C'2 by the deletion of thefive CH segments. This figure was adapted from [8,10].

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species are fed into the reactor and pass through the hot zone, where hydrocarbon precursors decompose to carbon radicals at metal substrate surface and then, form single-layer and few-layers. By the time the reaction occurs, the metal substrate not only works as a catalyst to lower the energy barrier to the reaction, but also determines the grapheme deposition mechanism.

CVD synthesis of Graphene on Cu

A large variety of metal substrates (Ni, Cu, Ru, Ir, Pt, Co, Pd and Re) can be used for grapheme growth. However, these metals show different carbon solubility and catalytic effect. In particular, single-layer grapheme growth on polycrystalline Cu films (Ruoff’s method) with a good control of grapheme layers, low cost, and ability to transfer. In this growth method, grapheme films were grown on 25 micrometre thick Cu foils in hot wall furnace. Initially, Cu foil was first annealed in hydrogen atmosphere at 1000 C, and then a mixture of H2/CH4 was introduced into the system to initiate the graphene growth. After a continuous graphene layer was formed on Cu foil, the system was cooled down to room temperature, with results shown in Figure 4.

Fig. 4. a) SEM image of graphene on a copper foil with a growth time of 30 min. (b) High-resolution SEM image of grapheme on Cu. Graphene films transferred onto a SiO2/Si substrate.(c) and a glass plate (d)

The Cu surface steps are formed during thermal annealing, and the darker flakes indicate multiple-layer graphene. Graphene “wrinkles” originate from the different thermal expansion coefficient of graphene and Cu. Those wrinkles can go across Cu grain boundaries, as Figure 4b shows, indicating that the graphene film is continuous. Graphene grown on Cu foil can be easily transferred to other substrates such as SiO2/Si and glass (Figure 3c, d) for further evaluation. The optical image analysis over a 1 1 cm2 region shows predominately monolayer graphene (>95%) with small fractions of bilayer (∼3 to 4%) and few-layer (<1%) graphene areas, which is confirmed by Raman spectra. Compared with other methods ( especially graphene growth on Ni), growth parameters such as film thickness and cooling rate have little influence on graphene CVD growth on Cu. Cu has ultralow carbon solubility. Even if the hydrocarbon concentration is high or the growth time is long, there is only a small amount of carbon dissolved in Cu. Most of the carbon source for graphene formation is from the hydrocarbon that is catalytically decomposed on the Cu surface (Figure 5). After the first layer graphene is deposited, Cu surface is fully covered and there is no catalyst exposed to hydrocarbon to promote decomposition and growth. Thus, the graphene growth on Cu is a surface reaction process, which is self-limiting and robust. This mechanism is experimentally proved by using isotopic labeling of hydrocarbon precursors combined with Raman spectroscopic mapping. Recently, various shapes of single-crystal graphene domains such as hexagonal, rectangular, and flower shape with different domain sizes have been achieved by varying growth parameters such as the methane con- centration and

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2. CVD SYNTHESIS AND GROWTH OF GRAPHENE

There exist comprehensive review articles and books dealing with generic aspects of the Chemical Vapor Deposition. Here we are only intent on describing elemental details of CVD applied to graphene synthesis. We encourage the readers to increase their knowledge with the fundamental aspects of Vapor Deposition Processes revisiting the cited references [17-23]. 2.1. BRIEF DESCRIPTION OF CVD CHEMICAL REACTIONS AND PROCESSES

Chemical Vapor Deposition (CVD) involves the activation of gaseous reactants and the subsequent chemical reaction followed by the formation of a stable solid deposit over a suitable substrate. The energy that the chemical reaction demands can be supplied with the aid of different sources; heat, light or electric discharge are used in thermal, laser assisted or plasma assisted CVD, respectively. The deposition process can include two types of reactions: homogeneous gas phase reactions, which occur in the gas phase, and heterogeneous chemical reactions which occur on/near the vicinity of a heated surface leading to the formation of powders or films, in each case. Though CVD has been used to produce ultrafine powders, this review article is mainly concerned with the CVD of extremely thin graphene films. So heterogeneous chemical reactions should be favoured and homogeneous chemical reactions avoided during the designed experiments. Fig. 1 shows a schematic diagram of a typical CVD process.

1. Transport of reactants by forced convection. 2. Thermal a) or plasma b) activation. Homogeneous gas reaction with particles and powder production should be avoided in graphene synthesis, controlling the kinetic parameters (P,T,n). 3. Transport of reactants by gas diffusion from the main gas stream through the boundary layer. 4. Adsorption of reactants on the substrate surface. 5. Dissolution and bulk diffusion of species depending on the solubility and physical properties of the substrate 6. Thermal activation mediated-surface processes, including chemical decomposition (catalytic), reaction, surface migration to attachment sites (such as atomic-level steps), incorporation and other heterogeneous surface reactions. Growth of the film. 7. Desorption of byproducts from the surface. 8. Transport of byproducts by diffusion through the boundary layer and back to the main gas stream. 9. Transport of byproducts by forced convection away from the deposition region.

Fig.1. Schematic diagram of Thermal CVD a) and Plasma Assisted CVD b) process: case of graphene from CH4/H2 mixtures.

a) b)

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a rough surface with many dark grains which indicate multi-ple graphene layers on polycrystalline Ni. Further character-ization usingmicro-Raman surfacemapping reveals that thearea percentage of monolayer/bilayer graphene for Ni(111)substrate is 91.4% (Figure 2c), much higher than the percen-tage of 72.8% for polycrystalline Ni (Figure 2f).

CVD Synthesis of Graphene on CuBesides nickel, people tried a variety of metal substratessuch as Cu,21 Ru,22 Ir,23 Pt,24 Co,25,30 Pd,26 andRe,31 showingdifferent carbon solubility and catalytic effect. In particular,the original study of high quality single-layer graphene growthonpolycrystalline Cu films21 reported by Ruoff et al. attracted alot of attention due to advantages such as good control ofgraphene layers, low cost, and ability to transfer.

In the growthmethod of Ruoff et al., graphene films weregrown on 25 μm thick Cu foils in a hot wall furnace.21

Initially, Cu foil was first annealed in hydrogen atmosphereat 1000 !C, and then a mixture of H2/CH4 was introducedinto the system to initiate the graphene growth. After acontinuous graphene layer was formed on Cu foil, thesystem was cooled down to room temperature, with resultsshown in Figure 3 (from ref 21). In Figure 3a, a low magni-fication SEM image of graphene on a copper substrateclearly shows the Cu grains with color contrast. More detailsof graphene morphology are revealed in the higher-resolu-tion SEM image (Figure 3b). The Cu surface steps are formedduring thermal annealing, and the darker flakes indicatemultiple-layer graphene. Graphene “wrinkles” originate

from the different thermal expansion coefficient of gra-phene and Cu. Those wrinkles can go across Cu grainboundaries, as Figure 3b shows, indicating that the gra-phene film is continuous. Graphene grown on Cu foil canbe easily transferred to other substrates such as SiO2/Si andglass (Figure 3c, d) for further evaluation. The optical imageanalysis over a 1 ! 1 cm2 region shows predominatelymonolayer graphene (>95%) with small fractions of bilayer(∼3 to 4%) and few-layer (<1%) graphene areas, which isconfirmed by Raman spectra.

FIGURE2. Schematic diagramsof graphenegrowthmechanismonNi(111) (a) and polycrystallineNi surface (d). Optical imageof graphenegrownonNi(111) (b) and polycrystalline Ni (e). Maps of IG0/IG of Raman spectra collected on the Ni(111) surface (c) and on the polycrystalline Ni surface (f)(Adapted with permission from ref 29. Copyright 2010 American Chemical Society).

FIGURE3. (a) SEM image of graphene on a copper foil with growth timeof 30 min. (b) High-resolution SEM image of graphene on Cu. Graphenefilms transferred onto a SiO2/Si substrate (c) and a glass plate (d).(Adapted from ref 21.)

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growth pressure. Some of the growth recipes are summarized in Table 1.

Fig. 4. Schematic diagram of grapheme growth mechanism on Cu.

Table 1. Graphene CVD on Cu films with different morphologies

8. GRAPHENE FILM TRANSFER

To facilitate graphene for nanoelectronic or photovoltaic applications, we need to remove the catalytic metal substrates from graphene and transfer graphene onto arbitrary substrates. A schematic diagram of the transfer process is shown in Figure 5. Graphene was first coated by a thin layer of polymethyl methacrylate (PMMA) and then baked at 120 °C to evaporate the solvent. The metal layer was then removed by Cu etchant, leaving only the PMMA/ graphene film. The film is cleaned by deionized (DI) water and then transferred onto a targeting substrate. After evap- orating water vapor away, PMMA was removed by acetone, leaving a graphene film on top of the targeting substrate. We were able to transfer both full wafer graphene from Ni film (Figure 5b left) and Cu foil using the above mentioned method onto glass, Si/SiO2 substrates and polyethylene terephthalate (PET) films.

Fig. 5. Graphene film transfer process.

9. GRAPHENE FUNCTIONALIZATION

Carboxyphenyl functionalization of the CVD graphene electrode surface is used to build the immunosensor. As shown inFig. 6. the 4-carboxyphenyl diazonium salt was prepared in-situ by mixing 2 mM sodium nitrite solution with 2 mM 4-aminobenzoic acid in 0.5 M HCl. The mixture was then stirred for 5 min at room temperature. The functionalization of CVD graphene electrodes (GE) was then performed by electroreduction of the carboxyphenyl diazonium cations using three cyclic voltammetric scans from +0.3 to −0.6 Vatascanrate of 100mVs-1.The electrode was then thoroughly rinsed with Milli-Q water. The 4-carboxyphenyl (CP) modified graphene electrode was activated by incubating the electrodes in MES buffer (pH 5.0) containing 100 mM EDC and 20 mM NHS for 60 min. After washing with MES buffer, the modified electrode surface was covered with anti OVA-MAb solution (10 µg mL-1 in 10 mM PBS buffer, pH 7.4) and incubated for 2 hours at room temperature in water-saturated atmosphere. The removal of the unreacted antibodies was performed by gently washing the electrodes with PBS buffer (10 mM, pH 7.4). Then, the electrodes were incubated in 0.1 % of BSA in PBS buffer, pH 7.4 for 30 min to deactivate the remaining active groups and to block the free graphene surface in order to minimize nonspecific adsorption. The prepared immunosensor was then rinsed with PBS and used in the assay or kept at 4◦C in PBS buffer pH 7.4 for later use.

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In our group, we carried out comparative study of gra-phene growth on Ni and Cu. Compared with graphenegrowth on Ni, growth parameters such as film thicknessand cooling rate have little influence on graphene CVDgrowth on Cu. The detail comparison is demonstrated inFigure 4. From the optical image of graphene transferredonto SiO2/Si substrate (Figure 4b), it is obvious that graphenegrown on polycrystalline Ni has many multilayer flakeswhile graphene on polycrystalline Cu is uniform monolayer(Figure 4e), which is also confirmed using Raman spectros-copy (Figure 4c and f). This significant difference suggestsdifferent graphene growth mechanisms on Cu and Ni.

For graphene formation on Ni, the growth mechanismhas been suggested to be a segregation process (Figure 4a),leading to difficulty in suppression of multilayer formation.In contrast, Cu has ultralow carbon solubility. Even if thehydrocarbon concentration is high or the growth time islong, there is only a small amount of carbon dissolved in Cu.Most of the carbon source for graphene formation is fromthe hydrocarbon that is catalytically decomposed on the Cusurface (Figure 4d). After the first layer graphene is deposited,Cu surface is fully covered and there is no catalyst exposed tohydrocarbon to promote decomposition and growth. Thus,the graphene growth on Cu is a surface reaction process,which is self-limiting and robust. This mechanism is experi-mentally proved by using isotopic labeling of hydrocarbonprecursors combined with Raman spectroscopic mapping.32

Recently, various shapes of single-crystal graphene do-mains such as hexagonal,33!37 rectangular,38,39 and flower

shape40!42 with different domain sizes have been achievedby varying growth parameters such as the methane con-centration and growth pressure. Some of the growth recipesare summarized in Table 2.

Transfer of Graphene FilmsTo facilitate graphene for nanoelectronic or photovoltaicapplications, we need to remove the catalytic metal sub-strates from graphene and transfer graphene onto arbitrarysubstrates. A schematic diagram of the transfer process isshown in Figure 5a. Graphenewas first coated by a thin layerof polymethyl methacrylate (PMMA) and then baked at120 !C to evaporate the solvent. The metal layer was thenremoved by Ni or Cu etchant, leaving only the PMMA/graphene film. The film is cleaned by deionized (DI) waterand then transferred onto a targeting substrate. After evap-orating water vapor away, PMMAwas removed by acetone,leaving agraphene filmon topof the targeting substrate.Wewere able to transfer both full wafer graphene from Nifilm (Figure 5b left) and Cu foil (Figure 5b right) using theabove-mentioned method onto glass (Figure 5c), Si/SiO2

substrates (Figure 5d), and polyethylene terephthalate(PET) films (Figure 5e).

Graphene Photovoltaic CellsGraphene films are transparent, conductive, and highlyflexible, which are considered to be great candidates fortransparent conductive electrodes in photovoltaic cells. TheconventionalOPV cells typically use indium tinoxide (ITO) as

FIGURE 4. Schematic diagrams of graphene growth mechanism on Ni (a) and Cu (d). Optical images of graphene transferred to SiO2/Si substratesfrom Ni substrate (b) and Cu substrate (e). Raman spectra collected on graphene synthesized using Ni (c) and Cu (f) substrates.

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anode materials, due to its extremely low sheet resistance(∼25 Ω/sq) and high transparency. However, the scarcityof indium reserves and highly brittle nature of metaloxides43,44 impose serious limitations on the use of ITO for

applications where cost, physical conformation, and me-chanical flexibility are important.45 CVD graphene films arehighly scalable, transparent (98% transparency for a single-layer graphene film), conductive (hundredsΩ to kΩ/square),

TABLE 2. Graphene CVD Recipes on Cu Films with Different Morphologies

ref annealing condition growth condition graphene morphology graphene grain size

21 1000 !C in2 sccm H2 at40 mTorr for 20 min

CH4 = 35 sccm, H2 = 2 sccm,pressure = 500 mTorr, time = 30 min,temperature = 1000 !C

continuous

33, 34 1050 !C in10 sccm H2 and300 sccm Arfor 30 min

50 ppm CH4 in Ar = 300 sccm,H2 = 20 sccm, pressure = 1 atm,time = 20 min,temperature = 1050 !C

hexagonal structure !18 μm

39 1045 !C in 50 sccm H2and 300 sccm Ar for 3h

CH4 =0.5 sccm, H2 = 500 sccm,pressure = 1 atm,time = 15 min,temperature = 1045 !C

regular square withsome jagged edges

up to 0.4 " 0.4 mm2

40 CH4 = 0.5 sccm and 1.3 sccm,partial pressure = 8 mTorrand 21 mTorr,H2 = 2 sccm, partialpressure = 27 mTorr,background pressure = 17 mTorr,time = 90 min, temperature = 1035 !Cwith Cu enclosure

hexagonal symmetryf six-sidedpolygons f very largegraphene domains with growingedges resembling dendrites

up to 0.5 mm

42 1000 !C in 7 sccmH2at 40 mTorr for 20 min

CH4 = 2 sccm, H2 = 25 sccm,pressure = 200 mTorr,time =30 min, temperature = 1000 !Cwith vapor trapping tube

predominantly six-lobe flowerswith four-lobe flowers insome location

∼100 μm

FIGURE 5. (a) Schematic diagram of the transfer process. (b) Wafer-scale synthesis of graphene on evaporated Ni film (left) and Cu foil (right).Graphene films were transferred onto a glass wafer (c), Si/SiO2 with device patterned (d), and a PET film (e).

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anode materials, due to its extremely low sheet resistance(∼25 Ω/sq) and high transparency. However, the scarcityof indium reserves and highly brittle nature of metaloxides43,44 impose serious limitations on the use of ITO for

applications where cost, physical conformation, and me-chanical flexibility are important.45 CVD graphene films arehighly scalable, transparent (98% transparency for a single-layer graphene film), conductive (hundredsΩ to kΩ/square),

TABLE 2. Graphene CVD Recipes on Cu Films with Different Morphologies

ref annealing condition growth condition graphene morphology graphene grain size

21 1000 !C in2 sccm H2 at40 mTorr for 20 min

CH4 = 35 sccm, H2 = 2 sccm,pressure = 500 mTorr, time = 30 min,temperature = 1000 !C

continuous

33, 34 1050 !C in10 sccm H2 and300 sccm Arfor 30 min

50 ppm CH4 in Ar = 300 sccm,H2 = 20 sccm, pressure = 1 atm,time = 20 min,temperature = 1050 !C

hexagonal structure !18 μm

39 1045 !C in 50 sccm H2and 300 sccm Ar for 3h

CH4 =0.5 sccm, H2 = 500 sccm,pressure = 1 atm,time = 15 min,temperature = 1045 !C

regular square withsome jagged edges

up to 0.4 " 0.4 mm2

40 CH4 = 0.5 sccm and 1.3 sccm,partial pressure = 8 mTorrand 21 mTorr,H2 = 2 sccm, partialpressure = 27 mTorr,background pressure = 17 mTorr,time = 90 min, temperature = 1035 !Cwith Cu enclosure

hexagonal symmetryf six-sidedpolygons f very largegraphene domains with growingedges resembling dendrites

up to 0.5 mm

42 1000 !C in 7 sccmH2at 40 mTorr for 20 min

CH4 = 2 sccm, H2 = 25 sccm,pressure = 200 mTorr,time =30 min, temperature = 1000 !Cwith vapor trapping tube

predominantly six-lobe flowerswith four-lobe flowers insome location

∼100 μm

FIGURE 5. (a) Schematic diagram of the transfer process. (b) Wafer-scale synthesis of graphene on evaporated Ni film (left) and Cu foil (right).Graphene films were transferred onto a glass wafer (c), Si/SiO2 with device patterned (d), and a PET film (e).

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4 Nano Res.

tube pressure of 1 Torr. Graphene growth was carried

out for 90 min, then, CH4 flow was cut off and the

tube was cooled down to room temperature while

maintaining H2 pressure at 700 mTorr. The graphene

grown on copper was then removed from the CVD

reactor.

2.4 Graphene transfer onto glass substrate

After growth, polymethyl methacrylate (PMMA) was

spin coated onto the surface of the graphene coated

Cu and baked at 120 oC for 1 min. The sample was

then immersed in ammonium persulfate (0.1 M)

solution to etch the Cu foil at room temperature. The

floating PMMA-supported graphene film was then

carefully transferred to a de-ionized water bath to

remove the residual etchant. The transfer of the

PMMA-graphene film to a clean DI water solution is

repeated at least three times to ensure thorough

washing. A glass sheet is then used to extract the

PMMA-supported graphene from the water bath.

The graphene sample was then dried at ambient

temperature for 48 h in a clean environment to

ensure complete adhesion to the glass. The PMMA

was then dissolved in a warm acetone bath to

produce a pristine graphene layer on glass sheet.

2.5 Connection of graphene sheet as working electrode

The graphene sheet was connected using conductive

tape and silver paste is used to improve the

connection between the copper and the graphene

sheet. A nonconductive adhesive polyimide tape was

then used to insulate the metal contacts and to

expose a well defined area of the graphene surface (3

×5 mm) to the solution during the electrochemical

measurements. The electrode was then connected to

the potentiostat through the copper contact.

2.6 Carboxyphenyl functionalization of the CVD graphene electrode surface and the immunosensor construction

As shown in Scheme 1, the 4-carboxyphenyl

diazonium salt was prepared in-situ by mixing 2 mM

sodium nitrite solution with 2 mM 4-aminobenzoic

acid in 0.5 M HCl. The mixture was then stirred for 5

min at room temperature. The functionalization of

CVD graphene electrodes (GE) was then performed

by electroreduction of the carboxyphenyl diazonium

cations using three cyclic voltammetric scans from

+0.3 to − 0.6 V at a scan rate of 100 mV s-1. The

electrode was then thoroughly rinsed with Milli-Q

water. The 4-carboxyphenyl (CP) modified graphene

electrode was activated by incubating the electrodes

in MES buffer (pH 5.0) containing 100 mM EDC and

20 mM NHS for 60 min. After washing with MES

buffer, the modified electrode surface was covered

with anti-OVA-MAb solution (10 µg mL-1 in 10 mM

PBS buffer, pH 7.4) and incubated for 2 hours at

room temperature in water-saturated atmosphere.

The removal of the unreacted antibodies was

performed by gently washing the electrodes with

PBS buffer (10 mM, pH 7.4). Then, the electrodes

were incubated in 0.1 % of BSA in PBS buffer, pH 7.4

for 30 min to deactivate the remaining active groups

and to block the free graphene surface in order to

minimize nonspecific adsorption. The prepared

immunosensor was then rinsed with PBS and used in

the assay or kept at 4◦C in PBS buffer pH 7.4 for later

use.

Scheme1 The electrografting process of carboxyphenyl groups on the graphene electrode and the subsequent antibody immobilization.

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Fig. 6. The electrografting process of carboxyphenyl groups on the graphene electrode and the subsequent antibody immobilization.

10. AUTOMATION

One of the first conditions for a machine to be efficient is to run alone, but have a good interaction between man and machine. This man-machine interface is also vital to the nanosensors. With an interface between the result of the analysis is captured by the nanosensor and a visible interface to man, becomes something essential for a good understanding of what is being studied. Being the ideal immediate interpretation by the result nanosensor, and the conversion of information, in which the results are refined by software database. Then, in this paper we can see a simple model of interface between man and machine, and too between analyte and man interface. Following the code made for the project, and a simple logic condition: by Pascal code:

Begin

program Checking analyte;

var

{ local variable definition }

a, analyte : integer;

begin

a := analyte;

if( a < 1 ) then

writeln(“there aren’t any infection agent in skin”)

else

writeln('detected material ' );

writeln('value of a is : ', a);

end.

11. THE HARDWARE

The hardware and system to support this project is a commercial technology, its need use flash memory to dispositive, like in images bellow.

Image 1.4 – Flash memory concept to project.

For the final design of this work, is ideal language C or C# that is accepted by the Arduino ™. To which it will interact and receive signals from the sensor, and a nanosensor that will capture the analysed information. There are programming a multitude of applications with advanced logic to process and work with data and information. Hence, in this paper was used one of the simplest conditions existing programming logic. As we can see in the following diagram:

Programming is a logical condition that allows him to interact with information captured by the sensor, and insert logic and work with information, making comparisons with other results. In addition, even using mathematics with statistics and other advanced procedures for working with data to indicate quantity or type or infectious agent in a sticker for (leprosys or other infection agent) in nanosensor. The program for reading and interpretation of the data get if analysis is detected, was done in a simple way, as showed before and the electrical signal send to software with statistical program to interpretation system, which in only a small window is shown. In the first line picks up the serial port, then check if

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the USB is connected to the Arduino or other hardware like a cellular and the nanosensor. The result is displayed in accordance with the quantity, level or energetic capacity of dust and the range of the nanosensor line.

The font code for this program is showed below:

Microsoft Visual Studio Solution File, Format Version 12.00

# Visual Studio 2013

VisualStudioVersion = 12.0.21005.1

MinimumVisualStudioVersion = 10.0.40219.1

Project("{FAE04EC0-301F-11D3-BF4B-00C04F79EFBC}") = "Readanalytesticker", " Readanalytesticker \ Readanalytesticker.csproj", "{EBA50ED7-38FA-443C-ACD8-AE5BCFDD5713}"

EndProject

Global

GlobalSection(SolutionConfigurationPlatforms) = preSolution

Debug|Any CPU = Debug|Any CPU

Release|Any CPU = Release|Any CPU

EndGlobalSection

GlobalSection(ProjectConfigurationPlatforms) = postSolution

{EBA50ED7-38FA-443C-ACD8-AE5BCFDD5713}.Debug|Any CPU.ActiveCfg = Debug|Any CPU

{EBA50ED7-38FA-443C-ACD8-AE5BCFDD5713}.Debug|Any CPU.Build.0 = Debug|Any CPU

{EBA50ED7-38FA-443C-ACD8-AE5BCFDD5713}.Release|Any CPU.ActiveCfg = Release|Any CPU

{EBA50ED7-38FA-443C-ACD8-AE5BCFDD5713}.Release|Any CPU.Build.0 = Release|Any CPU

EndGlobalSection

GlobalSection(SolutionProperties) = preSolution

HideSolutionNode = FALSE

EndGlobalSection

EndGlobal

As is clear, this design functions as a template being adaptable to any type of nanosensor, which can be a micro or nanosensor, which can be adjusted for reading physical, chemical, biological, or electrical. Simply by minor adjustments to your reading, interpretation and refinement of data collected.

The 1º nanosensor concept non-invasive

The project base is a portable apparel to make self-analyse and show immediately the results to user. The idea is showed in image 1.

Image 1 – Apparel to read if the person has or not disease by skin contact using stickers.

Image 2 – preview of the device for extern use, non-invasive check by contact if the person are contaminated. The nanosensor device could be made by a lot of antibodies, like a polymeric solution in wafer using as detector a biologic material named Interleukin 17 or I17 that is a biological natural material for detection of leprosys. The polymeric material is made of nanoplates in solution with I17 in an emulsion in leafs deposited by wafer architecture. This thin leafs are organized with switch keys and wires to capture electrical signals. This convert detection is converted in an electrical signal to

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hardware, a flash memory, and those to software programed that show the result in the display.

The idea is very simple; by a nanosensor system the use touch in they own skin and in seconds read in display the results. The person have or not the disease, the amount of bacily so this way it will be easier to choose its respective treatment (for paucibacillar or multybacillar) follow treatment. technology is based in nanowire where a leaf of nanowires functionalised capture analysis of the skin and detects the information, inside of 2 nm individual nanowires this touch is transformed in electrical signal, and sanded to a small hardware that interpret by a C function and show in display the results.

12. SKIN ADESION

In order to obtain an adhesion between human skin and device, it is necessary a polymer which has no chemical interaction whit electric circuit. The goal is achivied adopting gecko adhesion’s strategy. An array of micron-sized setae hairs offers geckos a unique ability to walk on vertical surfaces using Van der Waals interactions. This feature is can be used in order to attack a device as a sticker. In fact, each gecko toe (they have five per foot) contains thousands of microhairs, which are generally 100 microns long and five microns in diameter. Whereas the hairs on a gecko's toe are made from the protein keratin (a major component of human hair, skin and nails), some reasearcher made theirs from a polypropylene polymer. This gave them enough hardness to be packed together on a 0.4- by 0.8-inch (one- by two-centimeter) piece of white plastic they designed to simulate a gecko's toe. In this way, adhesive has similar adhesion to geckos, which can withstand a force of about 64.5 Newtons per square inch (10 Newtons per square centimeter).

13. CONCLUSION

This nanosensor will improve the humans with an additional human sense to monitoring it skin, but the purpose of this paper is show that knowing the process of

produces antibody and functionalized it, this turn possible adjusts to make any kind of human nanosensor. Nevertheless, this paper presents itself as a template for make artificial or synthetic antibodies.

14.References

[1].Graphene and graphene oxide: biofunctionalization and applications in biotechnology C2005 - Ying Wang et.al.

[2].Antibody phage display technology and its applications - C1998 - Hennie R. Hoogenboom

[3].Progress in Materials Science - C2010 - Chemical functionalization of graphene and its applications - Tapas Kuila et.al.

[4].Formulation Technology: Emulsions, Suspensions, Solid Forms - Hans Mollet, Arnold Grubenmann - WILEY-VCH Verlag GmbH, 2001

[5].Synthetic Peptides as Antigens - C2010 - for Antibody Production - David C. Hancock and Nicola J. O’Reilly

[6].Seminars in Cell & Developmental Biology - Antibody production, design and use for biosensor-based applications c2009 - Paul J. Conroy, Stephen Hearty, Paul Leonard, Richard J. O’Kennedy

[7].Methods - Synthetic antibodies: Concepts, potential and practical considerations - S. Miersch C2010, S.S. Sidhu - Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada.

[8] The role of surface chemistry in adhesion and wetting of gecko toe pads.- Badge I, Stark AY, Paoloni EL, Niewiarowski PH, Dhinojwala A, Sci Rep. 2014