Elsevier

Talanta

Volume 147, 15 January 2016, Pages 478-484
Talanta

Direct extraction of lead (II) from untreated human blood serum using restricted access carbon nanotubes and its determination by atomic absorption spectrometry

https://doi.org/10.1016/j.talanta.2015.10.023Get rights and content

Highlights

  • Metal bind sites of the restricted access carbon nanotubes (RACNTs) were mapped.

  • RACNTs were able to extract lead and to exclude all proteins from the samples.

  • Proteins were excluded by electrostatic repulsion.

  • A method was suitably validated for the lead analysis from human serum samples.

Abstract

Oxidized carbon nanotubes were covered with layers of bovine serum albumin to result in so-called restricted-access carbon nanotubes (RACNTs). This material can extract Pb2+ ions directly from untreated human blood serum while excluding all the serum proteins. The RACNTs have a protein exclusion capacity of almost 100% and a maximum Pb2+ adsorption capacity of 34.5 mg g−1. High resolution transmission electron microscopy, scanning transmission electron microscopy and energy dispersive spectroscopy were used to confirm the BSA layer and Pb2+ adsorption sites. A mini-column filled with RACNTs was used in an on-line solid phase extraction system coupled to a thermospray flame furnace atomic absorption spectrometry. At optimized experimental conditions, the method has a detection limit as low as 2.1 µg L−1, an enrichment factor of 5.5, and inter- and intra-day precisions (expressed as relative standard deviation) of <8.1%. Recoveries of the Pb2+ spiked samples ranged from 89.4% to 107.3% for the extraction from untreated human blood serum.

Introduction

New, sensitive and reliable methods for the analysis of low lead levels in biological samples can be important tools to identify and treat poisoning cases [1]. However, untreated biological samples cannot be directly analyzed by an analytical technique due to their complex matrix. In this case, a preparation step is needed in order to purify the sample, as well as make it compatible with the analytical techniques. First of all, the sample preparation procedures must be efficient to extract and preconcentrate the analytes, as well as to eliminate the interferents due to the high complexity of the biological samples (e.g. the presence of macromolecules), low concentrations of analytes and low sample volume availability. According to the literature, mineralization procedures using digestor blocks, ovens and/or microwave ovens are the most used methods for biological sample preparation of metal analyses [2], [3], [4]. Despite the wide use of these techniques, some drawbacks can also be pointed out, such as the need to high acidic concentrations, the possibility of analyte losses due to evaporation, as well as the slowness and complexity of the procedures, which can lead to low precision and accuracy [4].

Solid phase extraction (SPE) is an alternative to the mineralization procedures [5]. Some of the SPE advantages can be emphasized, like its easy cartridge/column regeneration, high analytical frequency, as well as high preconcentration factors for sorbents with high adsorption capacities, like carbon nanotubes (CNTs), which have been widely used for metal preconcentration [5], [6], [7], [8], [9]. Unmodified CNTs [10], [11], oxidized CNTs [12], [13], as well as CNTs modified with radical addition [14], [15] and composites [16], [17] are good examples of sorbents used in lead SPE. On the other hand, when the analytes have to be extracted from protein fluids (e.g. blood, plasma, serum), the high amount of proteins from the sample can be retained on the sorbent surface, obstructing the sorbent binding sites and leading to imprecision and inaccuracy.

Our research group developed a new material able to retain cadmium, and simultaneously exclude all proteins from biological fluids percolated through it [18]. These biocompatible particles, denominated restricted access carbon nanotubes (RACNTs), were obtained by the covering of commercial carbon nanotubes (CNTs) with bovine serum albumin (BSA) layers [18]. The BSA molecules were fixed on the CNT surfaces by interconnecting their amine groups, using glutaraldehyde as the cross linker reagent. Thus, when a biological fluid was percolated through a RACNTs' column, in a pH higher than the protein isoelectric point, both proteins from the sample and from the BSA layer were negatively charged, causing an electrostatic repulsion, responsible for the protein exclusion [18]. At the same time, the Cd2+ ions penetrated through the BSA layer, being retained in the CNTs' core [18]. The RACNTs were successfully used as a sorbent in an on-line SPE system to extract Cd2+ directly from untreated human blood serum samples, followed by thermospray flame furnace atomic absorption spectrometry (TS-FF-AAS) analyses. The samples were analyzed in the system without any previous treatment, demonstrating the high efficiency of the sorbent to purify complex matrices.

Based on the relevant characteristics of the RACNTs, as well as the importance of analyzing lead levels in blood serum samples, a new on-line SPE method for the extraction of Pb2+ ions, directly from these samples, followed by TS-FF-AAS analyses, was developed in this study. High resolution transmission electron microscopy was also used, scanning transmission electron microscopy and energy dispersive spectroscopy were used to investigate the exact metal adsorption sites in the RACNTs, complementing the previous characterization results presented in our first publication about RACNTs [18].

Section snippets

Reagents and solutions

All the reagents were of analytical grade and all the solutions were prepared with deionized water processed in Milli-Q system (Millipore, Bedford, USA). Multi-wall CNTs (with 6–9 nm of e.d.×5 µm of length) were purchased from Sigma Aldrich® (Steinheim, Germany). BSA, glutaraldehyde and sodium borohydride used in the CNT's coating were acquired from Sigma Aldrich. The Pb2+ standard solutions were prepared by dilutions of 1000 mg L−1 Pb2+ stock solution purchased from Sigma Aldrich. Nitric acid, as

Synthesis and characterization of the restricted access carbon nanotubes

TEM images of the CNTs and RACNTs (Fig. 2a–d) were obtained using two types of CCD cameras. A Gatan ES 500W CCD camera was used to acquire the image of the original CNTs (Fig. 2a) and the RACNTs (Fig. 2b). The CNTs were evolved in BSA capsules formed by the interconnection between the BSA amine groups, using glutaraldehyde as a cross-linker. The reaction resulted in imines that were reduced to amines using sodium borohydride. This procedure was necessary to give stability to the BSA layer [20],

Conclusion

The RACNTs presented high Pb2+ extraction capacity from human serum samples, without previous sample preparation steps. It was also confirmed that the metal ions penetrate through the BSA layer, being preferably retained in the CNT core. It was also possible to perform a full and fast protein exclusion in the present system. The RACNTs' column could be used for more than 200 sequential extractions of Pb2+, without losing the analyte extraction efficiency. The enrichment factor allowed the Pb2+

Acknowledgments

The authors are thankful to the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG, Belo Horizonte, Brazil), Projects CDS-PPM-00144-15 and CEX-APQ-01556-13; the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brasília, Brazil), Project 483371/2012-2; and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brasília, Brazil) for their financial support.

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