Carbon Nanohorns as a Scaffold for the Construction of Disposable

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Carbon nanohorns as scaffold for the construction of disposable electrochemical immunosensing platforms. Application to the determination of fibrinogen in human plasma and urine Irene Ojeda, Belit Garcinuño, María Moreno-Guzmán, Araceli González-Cortés, Masako Yudasaka, Sumio Iijima, Fernando Langa, Paloma Yanez-Sedeno, and Jose M. Pingarron Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac501681n • Publication Date (Web): 07 Jul 2014 Downloaded from http://pubs.acs.org on July 11, 2014

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Analytical Chemistry

Carbon nanohorns as scaffold for the construction of disposable electrochemical immunosensing platforms. Application to the determination of fibrinogen in human plasma and urine Irene Ojedaa, Belit Garcinuñoa, María Moreno-Guzmána, A. González-Cortésa, Masako Yudasakab , Sumio Iijimac, Fernando Langad, Paloma Yáñez-Sedeñoa, José M. Pingarróna* a

Department of Analytical Chemistry, Faculty of Chemistry, University Complutense of Madrid.

28040-Madrid. Spain. b Nanotube Research Center, National Institute of Advanced Industrial and Technology, Higashi, Tsukuba, Ibaraki 305-8565. Japan.

c

Department of Physics, Meijo

University, Shiogamaguchi, Tenpaku-ku, Nagoya 468-8502. Japan.

d

Instituto de Nanociencia,

Nanotecnología y Materiales Moleculares (INAMOL), Universidad de Castilla-La Mancha, 45071-Toledo. Spain.

*Corresponding author. Fax: +34913944329. E-mail: [email protected] ABSTRACT. We describe in this work a novel electrochemical immunosensor design making use of carbon nanohorns (CNHs) as scaffold for the preparation of disposable immunosensing platforms for the determination of fibrinogen (Fib). The approach involved the immobilization of

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Fib onto activated CNHs deposited on screen-printed carbon electrodes and the implementation of an indirect competitive assay using anti-Fib labeled with horseradish peroxidase (HRP) and hydroquinone (HQ) as the redox mediator. Both CNHs and the Fib-CNHs covalent assemble were characterized by microscopic and electrochemical techniques. The different variables affecting the analytical performance of the amperometric immunosensing strategy were optimized. The calibration plot for Fib allowed a range of linearity between 0.1 and 100 µg/mL (r2 = 0.994) and a detection limit of 58 ng/mL to be achieved. The Fib-CNHs/SPCEs exhibited an excellent storage stability of at least 42 days. The developed immunosensor provides, in general, a better analytical performance than that reported for other Fib immunosensors and commercial ELISA kits. This simple and relatively low cost immunosensor configuration permitted the sensitive and selective determination of Fib in human plasma and urine.

KEYWORDS. carbon nanohorns, electrochemical immunosensor, fibrinogen.

INTRODUCTION Carbon nanohorns (CNHs) consist of single-wall graphene sheets having tubule structure with diameters of 2-5 nm and length 40-50 nm with conical-shaped tips. Several thousand CNHs assemble to form quasi-spherical aggregates shaped like a dahlia flower. CNHs are produced by laser ablation of pure graphite in absence of metallic catalysts1. The spherical aggregate structure enabled the easy dispersion in liquids and the absence of metals affords to use them without metal removal purification. These advantages of CNHs over the other nanocarbons have promoted interest in the use of this material in various applications2,3. CNHs with pseudocylindrical forms have bumps and dips where existing pentagonal and heptagonal rings4. The


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number of structure defects was larger than those of straight-shaped carbon nanotubes. Such defects are easily oxidized and holes are opened4, generating abundant oxygenated groups5, which is also attractive to various applications. By opening hole, the inner space becomes accessible, and the specific surface area is enlarged from about 300 m2/g to 1400 m2/g6 . CNHs-modified electrodes have been prepared to observe direct electron transfer from proteins. For example, glucose oxidase (GOx) immobilized onto a Nafion-CNHs film displayed cyclic voltammograms showing a pair of redox peaks at a formal potential of -0.453 V. A GOx biosensor based on this configuration was prepared for the determination of glucose in a linear range from 0 to 6.0 mM using ferrocene monocarboxylic acid as the redox mediator7. More recently, the same authors prepared a CNHs film functionalized non-covalently with poly(sodium 4-styrenesulfonate) for the immobilization of myoglobin, and reported the fabrication of an electrochemical biosensor showing direct electrochemistry and electrocatalytic ability towards the reduction of hydrogen peroxide8. Direct electron transfer of soybean peroxidase (SBP) adsorbed on CNHs-modified electrodes along with effective electrocatalysis on the reduction of H2O2 were also observed9. Moreover, a biosensor using synthesized gold nanoparticles/CNHs hybrids and xanthine oxidase immobilized on a platinum electrode was applied to the detection of hypoxanthine and xanthine in a fish sample10. Regarding the use of CNHs for the preparation of electrochemical immunosensors, only two configurations were found in the literature. A design was proposed for the determination of microcystin-LR (MC-LR) involving covalent immobilization of the antigen to oxidized CNHs and the use of horseradish peroxidase-labeled MC-LR antibody for the development of a competitive immunoassay11. Very recently, a sandwich-type impedimetric immunosensor based on linking HRP and GOx enzymes to CNHs and biocatalyzed precipitation was reported for the


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detection of α-fetoprotein (AFP). The enzymes accelerated the oxidation of 4-chloro-1-naphthol by H2O2 to yield an insoluble product on the electrode surface which led to a significant enhancement of the signal from Fe(CN)63-/4- redox couple12. In an attempt to go beyond the use of CNHs as scaffold for the preparation of electrochemical immunosensing platforms, we describe in this work a novel design which was applied to the determination of fibrinogen (Fib). The approach involved the immobilization of Fib onto activated CNHs deposited on disposable screen-printed carbon electrodes (SPCEs), and the implementation of an indirect competitive assay using anti-Fib labeled with horseradish peroxidase (HRP) and hydroquinone (HQ) as the redox mediator. This simple and relatively low cost immunosensor configuration permitted the sensitive and selective determination of Fib in human plasma and urine. Fib is a plasmatic glycoprotein produced by the liver that plays a key role in the hemostatic system. Normal levels of Fib in human plasma range between 1.5 and 4.5 mg/mL13. Lower concentrations indicate the risk of bleeding and may be related to liver diseases, whereas higher Fib levels are associated with cardiovascular diseases14,15. Plasmatic Fib can also be used as a biomarker for metabolic syndrome16 and gastric and ovarian cancer17,18. The determination of Fib and Fib degradation products in urine has also a great interest for the detection of nephrotic syndrome19 and bladder cancer20,21. Measurement of Fib in clinical samples has been performed using a variety of methodologies. Apart from the early procedures based on the clotting time assessment22 or the prothrombin timederived assay23, techniques such as immuno-nephelometry24 or turbidimetry25 have been used. Based on the precipitation of fibrin, a point of care lateral flow device was developed using a thrombin reagent that made Fib non soluble. As a result of clotting, lateral sample flow is


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detained, and the distance along the device at which this occurs depends on the fibrinogen concentration15. Regarding biosensors, a disposable design was described based on a thrombin co-substrate which is competitive with Fib for thrombin. The enzyme hydrolysis of the substrate releases an electroactive compound so that the amperometric measurement allows a method for Fib to be proposed with a linear range between 1 and 3 mg/mL26. Moreover, a SPR biosensor using the principle of competitive adsorption of proteins was also reported27. Pre-adsorbed IgM on the sensing surface was selectively displaced by Fib this giving rise to a refractive index change recorded as the angle shift which is proportional to Fib concentration between 0.5 and 2.5 mg/mL. Furthermore, very recently, disposable amperometric magnetoimmunosensors using nanobodies as biorecognition element were prepared by our group and applied to the determination of Fib in human plasma28.

EXPERIMENTAL SECTION Reagents and solutions Fibrinogen (Fib) from human plasma was from Sigma. Mouse monoclonal anti-Fib antibody labeled with HRP (HRP-anti-Fib) was from HyTest. CNHs with a >90 % purity were produced by CO2 laser ablation of graphite in the absence of any metal catalyst under an argon atmosphere (760 Torr) at room temperature (the 10% impurity is micrometer-sized graphitic balls29). Carboxylic acid groups were generated on CNHs by treatment with H2O2 aqueous solution at about 100ºC5. Hydroquinone (HQ), hydrogen peroxide (30%, w/v), N-(3-dimethyl aminopropyl)N’-ethylcarbodiimide (EDC) and N-hydroxysulfosuccinimide (Sulfo-NHS) were from Sigma-


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Aldrich. EDC/Sulfo-NHS mixture solutions (100 mM each in MES buffer, pH 5.0) were used for activation of the carboxylated CNHs. Sodium di-hydrogen phosphate and di-sodium hydrogen phosphate were from Scharlau. 2-(N-morpholine) ethanesulfonic acid (MES) and bovine serum albumin (BSA) were from GERBU Biotechnik, GmbH. Casein from bovine milk (Sigma) and semi-skimmed milk purchased in a local supermarket were also used. Buffer solutions used were 0.025 M MES buffer pH 5.0, and phosphate buffer solutions of pH 7.4 (PBS), and 6.0. Human hemoglobin (Hb, H7379), IgG from human serum (I2511), both from Sigma, and D-dimer (Abcam, ab35949), were tested as potential interfering substances. Deionized water was obtained from a Millipore Milli-Q purification system (18.2 MΩ cm).

Apparatus and electrodes All electrochemical measurements were carried out with a PGSTAT 12 potentiostat from Autolab. The electrochemical software was the general-purpose electrochemical system (GPES) (EcoChemie B.V.). The screen printed carbon electrodes (SPCEs, 110 DRP, 4 mm ∅) and the carboxylated-MWCNTs modified-SPCEs (110 CNT DRP, 4 mm ∅) were purchased from DropSens (Oviedo, Spain). These electrodes include a silver pseudoreference electrode and a carbon counter electrode. SPCEs modified with carboxylated CNHs were used as the working electrodes. All experiments were performed at room temperature.

Samples


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A WHO 3rd International Standard for Fib Plasma (National Institute for Biological Standards and Control, NIBSC, code: 09/264, UK), containing 2.7 mg/mL Fib was analyzed. Spiked urine samples were from Liquichek Urine Chemistry Control (Level 1, BioRad 63221).

Procedures Preparation of HRP-anti-Fib-Fib-CNHs/SPCEs immunosensors CNHs/SPCEs were prepared by casting 10 µL of a 0.5 mg carboxylated CNHs per mL aqueous dispersion onto the electrode surface and drying under IR radiation. Then, 10 µL of a 100 mM EDC/Sulfo-NHS solution were dropped onto the CNHs/SPCE and left to stand for 30 min. After washing with MES buffer solution of pH 5.0, 10 µL of a 25 µg/mL Fib solution were dropped onto the electrode surface and kept to dryness. Then, a blocking step of the remaining unmodified electrode sites was accomplished by adding 10 µL of semi-skimmed milk 1/4 diluted in phosphate buffer solution of pH 7.4 and allowing incubation for 30 min. In order to perform the indirect competitive immunoassay, 10 µL of a mixture solution containing 1.0 µg/mL HRP-anti-Fib and the Fib standard solution or the sample in 0.1 M PBS of pH 7.4, were dropped onto the electrode surface and incubated for 30 min. Fib determination was accomplished by dropping 45 µL of a 1 mM HQ solution prepared in 0.05 M phosphate buffer solution of pH 6.0 onto the surface of the horizontally positioned HRP-anti-Fib-Fib-CNHs/SPCE immunosensor and by applying a potential of -0.05 V. When the background current was stabilized (less than 100 s), 5 µL of a 50 mM H2O2 solution prepared in the same buffer were added and allowed standing for 200 s, upon which the current produced by the electrochemical reduction of benzoquinone was measured.


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Determination of Fib in human plasma and urine According to the instructions recommended by the supplier of the certified plasma, the total content of the ampoule was reconstituted at room temperature with 1 mL of deionized water. Then, gentle swirling to avoid froth was applied until complete dissolution, and the sample was transferred immediately to a plastic tube. Fib determination was performed after an appropriate dilution of the reconstituted plasma in 0.1 M PBS of pH 7.4. Urine samples from Liquichek Urine Chemistry Control (Level 1, BioRad 63221), spiked with 0.65, 5.0 and 25.0 µg/mL Fib, were also analyzed. In both cases, no matrix effects were observed, and, consequently, the amperometric responses from the samples were directly interpolated into the calibration plot constructed with Fib standard solutions.

RESULTS AND DISCUSSION Characterization of CNHs and Fib-CNHs As it is described in the Experimental section, carboxylic acid groups were generated on CNHs by treatment with H2O229-31. This mild oxidation procedure opened holes on the tips of CNHs incorporating carboxylic groups needed for further functionalization. It has been reported that, comparing with the oxidation with air or oxygen gas, CNHs treated with H2O2 solution have larger number of –COOH groups. These groups were identified by IR and TG-MS measurements and the quantities were estimated by TGA performed in He29-31. Raman spectra of oxidized CNHs with H2O2 showed an increased D/G bands ratio with respect to pristine CNH which was attributed to the material functionalization31. Moreover, XPS analysis of the treated CNH showed an increase on the oxygen content due to the presence of the carboxylic groups32,33.


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Both carboxylated CNHs and the Fib-CNHs covalent assemble were also characterized by means of microscopic techniques. Figure 1A shows a transmission electron microscopy (TEM) image of carboxylated CNHs which exhibited the typical morphology of dahlia bundles with a diameter of about 120 nm. Once deposited on the surface of the SPCEs, the spherical structures were maintained and the mean diameter was around 130 nm as revealed by scanning electron microscopy (SEM) (Figure 1B), which demonstrated that the electrode surface deposition process did not significantly affect the original CNHs morphology. Furthermore, Figures 2A and 2B displays atomic force microscopy (AFM) images of pristine CNHs and Fib-CNHs conjugates prepared by covalently binding of fibrinogen to carboxyl groups of nanohorns. As it can be seen, highly dispersed CNHs appearing as small spherical structures in Figure 2A, whereas Fib-CNHs aggregates (Figure 2B) exhibited a much larger size, of about 450 nm, as a result of the biomolecule immobilization.


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Figure 1. (A) TEM image of carboxylated CNHs (scale bars, 50 nm); (B) SEM image from CNHs deposited onto a SPCEs.

Figure 2. AFM images of pristine CNHs (A) and Fib-CNHs conjugates (B). The different steps involved in the preparation of the Fib-CNHs/SPCE immunosensor were also characterized by electrochemical impedance spectroscopy (EIS). Figure 3 shows the Nyquist plots recorded in a 1 mM Fe(CN)64−/Fe(CN)63− solution in 0.1 M PBS of pH 7.4 for a bare SPCE and CNHs/SPCE, and Fib-CNHs/SPCE modified electrodes. As expected, the CNHs/SPCE exhibited the lowest charge transfer resistance (Rct), 80 Ω, which is a much smaller value than that at the bare SPCE, Rct=1680 Ω, showing a faster electron transfer at the CNHs-modified electrode. This result is in agreement with the corresponding cyclic voltammograms (inset of Fig. 3).


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Moreover, as expected, the immobilization of Fib on the CNHs/SPCE resulted in a remarkable increase of the Rct value, (800 Ω) as a result of the insulating character of the protein.

Figure 3. Nyquist plots recorded for a bare SPCE (black), CNHs/SPCE (grey) and FibCNHs/SPCE (white) in 1 mM Fe(CN)64−/Fe(CN)63− 0.1 M PBS of pH 7.4. Inset: cyclic voltammograms from SPCE (black) and CNHs/SPCE (grey) in 1 mM Fe(CN)64−/Fe(CN)63− 0.1 M PBS of pH 7.4; ν = 50 mV/s.

Design of the electrochemical immunosensing approach at the Fib-CNHs/SPCEs Figure 4 shows schematically the rational of the different steps involved in the immunosensing approach for Fib using Fib-CNHs/SPCE modified electrodes. This approach used an indirect competitive immunoassay which was chosen to shorten the whole procedure and economize immunoreagents which are important practical advantages for the development of useful clinical assays.


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Figure 4. Schematic display of the different steps involved in the amperometric immunosensing approach for the quantification of Fib using Fib-CNHs/SPCEs. Once the SPCE was modified with carboxylated CNHs (step 1) and Fib was covalently immobilized through carbodiimide chemistry (step 2), an indirect competitive immunoassay involving standard solutions of target Fib solutions (or the sample) and HRP-anti-Fib was carried out by drop casting an aliquot of a mixture solution of both immunoreagents onto the Fib-CNHs/SPCE and incubating for 30 min (step 3). The remaining free HRP-anti-Fib reacted with the immobilized Fib (step 4) and, thereafter, the target Fib was quantified amperometrically upon addition of hydrogen peroxide in the presence of hydroquinone as redox mediator (step 5).


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Optimization of the experimental variables involved in the performance of the electrochemical immunosensing approach The different variables affecting the analytical performance of the amperometric immunosensing strategy were optimized. The evaluated variables were: a) the CNHs loading on the SPCE; b) the Fib loading on the activated CNHs; c) the reagents and incubation time involved in the blocking of the remaining unmodified electrode sites; d) the concentration of HRP-anti-Fib, and the need of a pre-incubation period of HRP-anti-Fib and the target Fib in the mixture solution; e) the potential value employed in the amperometric detection. Details on these optimization studies can be found in the text and Figs. S1 to S4 in the Supporting Information. The range of the variables checked and the corresponding selected values are summarized in Table 1. Table 1. Variables optimized and selected values regarding the performance of the HRPanti-Fib-Fib-CNHs/SPCEs Variable CNHs loading, mg/mL Fib loading, µg/mL Blocking agent type Blocking agent, dilution ratio in PBS Incubation time for blocking, min HRP-anti-Fib, µg/mL Detection potential, mV

Tested range 0.25 - 1.0 17.5 - 75 casein, BSA, milk 1/10 - 1/1 15 - 60 0.25 - 2.0 0 to – 200

Selected value 0.5 25 milk ¼ 30 1.0 - 50

Analytical performance The calibration plot for Fib constructed with the HRP-anti-Fib-Fib-CNHs/SPCE immunosensor, prepared under the optimized working conditions summarized in Table 1, is shown in Figure 5. Error bars were calculated from the measurements carried out with three different immunosensors in each case. Some of the corresponding amperometric recordings are displayed


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in Figure S5 showing the expected shape for this type of amperometric measurements where only diffusion of the electroactive substrate occurred. The current vs. Fib concentration curve was fitted by non-linear regression using the Sigma-Plot data analysis software. The adjusted equation (r2 = 0.998) was: =

i − i

EC 1 + x

+ i

where imax = 2.02 ± 0.03 µA and imin = 0.13 ± 0.04 µA are the maximum and minimum current values in the calibration graph. The EC50 value, which corresponded to the Fib concentration producing 50% competition, was 2.5 ± 0.3 µg/mL, with a Hill slope, h, of 0.66 ± 0.05 µg/mL. The range of linearity extended between 0.1 and 100 µg/mL (r2 = 0.994), and followed the equation: i, µA = - (0.56±0.02) log ([Fib], µg/mL) + (1.32±0.02). This range is appropriate for application to plasma samples once they are diluted for at least 100 times which, as demonstrated below, removes potential matrix effects. The achieved limit of detection, 58 ng/mL, was calculated from the equation: %

$ & − = − 1# − − 3"

where s is the standard deviation (n = 5) of the zero value (i.e. the current value measured with no Fib, 46 nA). The reproducibility of the amperometric measurements was evaluated both without target Fib and in the presence of 2.5 µg/mL target Fib. Different immunosensors were prepared both on the same day and on different days to carry out the measurements and a new CNHs/SPCE was used in each case. The relative standard deviation (RSD) values obtained (n=8) were 3.8 % and 6.9 % for the assays performed on the same day in the absence and in the presence of target Fib, respectively, whereas RSD (n=8) values were 6.7 % and 7.3 %, respectively, for the measurements made on different days. These results revealed the good level


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of precision achieved considering the whole preparation process and the measurement protocol of the proposed nanostructured immunosensors.

Figure 5. Calibration plot constructed for Fib using amperometry at the HRP-anti-Fib-Fib CNHs/SPCE immunosensor. See text and Table 1 for the experimental conditions.

The storage stability of the Fib-CNHs/SPCE modified electrodes at -20 ºC under dry conditions was also evaluated. Various bioelectrodes were prepared on the same day, stored and used to construct the corresponding immunosensors in different days. Figure 6 displays the control chart constructed for the currents measured for solutions containing no Fib. As it can be seen, the immunosensor responses remained within the control limits, set at ± 2 times the standard deviation of the measurements (n = 3) carried out on the first day, for at least 42 days (no longer periods were checked) which indicated an excellent storage stability of the Fib-CNHs/SPCEs


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suggesting the possibility of their preparation and storage under the above specified conditions and the use for the immunosensors fabrication on request.

Figure 6. Control chart constructed to evaluate the storage stability of Fib-CNHs/SPCE bioelectrodes. Each point corresponds to the mean value of three successive measurements in the absence of target Fib.

Table 1 in Supporting Information compares the analytical characteristics provided by the developed immunosensor with those reported for other Fib immunosensors and ELISA kits. In particular,

when

the

comparison

is

made

against

the

previously

reported

magnetoimmunosensors28, it can be seen as the range of linearity covers practically the same range of Fib concentrations than that of the magnetoimmunosensor using a direct competitive immunoassay, but the limit of detection is more than five times lower and the time of analysis remarkably shorter. Regarding ELISA kits, it can be concluded that, in general, the developed immunosensor provides wider analytical linear range together with a better sensitivity, higher


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precision and shorter analysis time than those achieved with most of the commercial kits.The response of the CNHs-based immunosensor was compared with that observed by immobilizing Fib using the same approach on commercial carboxylated MWCNTs-SPCEs. Figure 7 displays histograms showing the measured specific-to-non specific (i.e. with no immobilized Fib) current ratios for three different target Fib concentrations at both CNHs/SPCE and MWCNTs/SPCE based immunosensors. As it can be easily observed, whereas a clear difference in the immunosensor response was observed when CNHs are involved in the bioelectrode construction (as expected considering that the target concentrations are within the linear range of the calibration graph) no sufficiently differentiated specific-to-nonspecific current ratios were apparent for the lower target Fib concentrations at the Fib-MWCNTs/SPCE. These results indicated that a better detection limit was achievable with the Fib-CNHs/SPCEs. This advantageous behavior had been suggested previously by several authors10,34,35. Among the most widely claimed reasons for this, the way in which CNHs are synthesized is prominent. The mild and short in time but very powerful oxidative treatment used introduces carboxylic units at the cone end of the dahlia-type aggregates avoiding shortening of CNHs and formation of any type of impurities, thus, retaining the CNHs high purity. This step greatly differentiates the whole functionalization procedure from the original one applied for the tip opening of CNTs (i.e. treatment under harsh conditions, e.g., strong acids, reflux, ultrasonication, and long times) which in addition cut the original long nanotubes in short pieces. Moreover, in contrast to strong acids used for creating the sites of chemical modification with CNTs, the use of H2O2, a milder oxidizing agent, is advantageous in controlling the degree of oxidation and for generating minimal amounts of carbonaceous by product36.


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Figure 7. Specific-to-non specific current ratios for: 0.1 µg/mL (black), 1 µg/mL (grey) and 5 µg/mL (white) target Fib at both CNHs/SPCE and MWCNTs/SPCE based immunosensors.

Selectivity Various proteins (hemoglobin (Hb), D-dimer, BSA, and IgG) were tested as potential interfering substances for the determination of Fib using the developed immunosensor. The evaluation of the selectivity was accomplished by comparing the immunosensor responses obtained for 0 and 2.5 µg/mL Fib in absence and in the presence of each tested compound. Figure 8 shows as not significantly different currents were measured in both cases in the presence of 500 ng/mL Hb, 500 µg/mL BSA or 47 µg/mL IgG. These concentrations corresponded to the usual concentration levels commonly found in plasma samples once these samples are 1/100 diluted. Moreover, a concentration of D-dimer six times higher than that expected in healthy individuals, did neither show a significant interference despite the similar sequence of amino acids to that of Fib37.


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Figure 8. Effect of the presence of hemoglobin (Hb), D-dimer, BSA and IgG on the amperometric responses obtained for 0 (grey) and 2.5 (black) µg/mL Fib at the HRP-anti-FibFib-CNHs/SPCE immunosensor.

Determination of Fib in human plasma and urine The HRP-anti-Fib-Fib-CNHs/SPCE immunosensor was validated by application to the analysis of an International Standard for Fib Plasma containing a certified concentration of 2.7 mg/mL Fib. Moreover, spiked urine samples at 0.65, 5.0 and 25.0 µg/mL Fib levels were also analyzed. The possible existence of matrix effects was investigated in both types of sample by comparing the slope values of the calibrations plots constructed for Fib over the 0.1 and 100 µg/mL range with Fib standard solutions and in the samples matrices. The corresponding regression equations were i, µA = -(0.58±0.01) log ([Fib], µg/mL) + 1.35±0.01 in plasma and i, µA = -(0.57±0.01) log ([Fib], µg/mL) + 1.34±0.01 in urine, revealing that no statistically significant differences, at a significance level of 0.05, existed between these slope values and that obtained with Fib standard


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solutions (-0.56±0.02 µA per decade of concentration). Therefore, the determination of Fib could be performed by direct interpolation of the amperometric values measured from the samples solutions into the calibration plot constructed with standard solutions. As it was mentioned in section 2.4.2, once reconstituted with 1 mL of deionized water, plasma was diluted in 0.1 M PBS of pH 7.4. A mean value of 2.7 ± 0.2 mg/mL Fib (n=8), with a RSD value of 7.4% was obtained which corresponded to a mean recovery of 99 ± 7%. In the case of urine analysis, solid phosphate salts were added to the spiked sample in order to adjust the initial pH of 5 to 7.4. The obtained Fib mean concentrations were 0.65 ± 0.05; 4.9 ± 0.4 and 24 ± 2 µg/mL, corresponding to recoveries ranging between 98 ± 8% and 100 ± 7%. All these results demonstrated the usefulness of the developed immunosensor for the analysis of Fib in human plasma and urine at the physiological levels that can be expected in clinical samples with practically no sample treatment.

CONCLUSIONS The obtained results show clearly that carbon nanohorns can be used as a complement and/or advantageous scaffold nanomaterial for improving electrochemical immunosensing properties. The unique structure and high purity of CNHs produce a large amount of oxygenated moieties that can be harnessed to the incorporation of biomolecules such as the immunoreagents used in this work. This superior behavior has been illustrated by preparing an amperometric disposable immunosensor for the determination of fibrinogen that exhibits an excellent analytical performance in terms of sensitivity, selectivity, reproducibility of the measurements and storage stability. This performance has demonstrated to be superior, in general, to that reported for other


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Fib immunosensors and commercial ELISA kits. Importantly, the Fib-CNHs/SPCE could be successfully applied to the rapid and accurate quantification of Fib in human plasma and urine at the physiological levels that can be expected in clinical samples with practically no sample treatment.

ACKNOWLEDGMENT Financial support from the Spanish Ministerio de Economía y Competitividad (Project CTQ2012-35041) is gratefully acknowledged.

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Figure 4. Schematic display of the different steps involved in the amperometric immunosensing approach for the quantification of Fib using Fib-CNHs/SPCEs. 254x190mm (96 x 96 DPI)


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Carbon Nanohorns as a Scaffold for the Construction of Disposable

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