Elsevier

Bioelectrochemistry

Volume 111, October 2016, Pages 7-14
Bioelectrochemistry

Human serum albumin-stabilized gold nanoclusters act as an electron transfer bridge supporting specific electrocatalysis of bilirubin useful for biosensing applications

https://doi.org/10.1016/j.bioelechem.2016.04.003Get rights and content

Highlights

  • The AuNCs formed in HSA act as an electron transfer bridge for bilirubin oxidation.

  • The oxidation occurs at a specific moiety of bilirubin on the electrode surface.

  • The HSA-AuNCs bioelectrode detects free bilirubin sensitively in serum samples.

Abstract

Human serum albumin (HSA)-stabilized Au18 nanoclusters (AuNCs) were synthesized and chemically immobilized on an Indium tin oxide (ITO) plate. The assembly process was characterized by advanced electrochemical and spectroscopic techniques. The bare ITO electrode generated three irreversible oxidation peaks, whereas the HSA-AuNC-modified electrode produced a pair of redox peaks for bilirubin at a formal potential of 0.27 V (vs. Ag/AgCl). However, the native HSA protein immobilized on the ITO electrode failed to produce any redox peak for bilirubin. The results indicate that the AuNCs present in HSA act as electron transfer bridge between bilirubin and the ITO plate. Docking studies of AuNC with HSA revealed that the best docked structure of the nanocluster is located around the vicinity of the bilirubin binding site, with an orientation that allows specific oxidation. When the HSA-AuNC-modified electrode was employed for the detection of bilirubin using chronoamperometry at 0.3 V (vs. Ag/AgCl), a steady–state current response against bilirubin in the range of 0.2 μM to 7 μM, with a sensitivity of 0.34 μA μM 1 and limit of detection of 86.32 nM at S/N 3, was obtained. The bioelectrode was successfully applied to measure the bilirubin content in spiked serum samples. The results indicate the feasibility of using HSA-AuNC as a biorecognition element for the detection of serum bilirubin levels using an electrochemical technique.

Introduction

Quantitative detection of the free bilirubin levels in blood serum has an enormous clinical importance in probing hyper bilirubinemia conditions, such as bilirubin encephalopathy [1], [2], [3], [4]. Among the various analytical methods [5], [6], peroxidase-based enzymatic methods are widely used to detect the unbound fraction of bilirubin [7], [8]. Although reliable, the aforementioned analytical methods are complex, time consuming, dependent on skilled operators, and require an expensive infrastructure. Global efforts have focused on developing biosensors that offer rapid, reliable, simple and economical detection of analytes for use in point of care set up [9]. Electrochemical sensors have received increasing attention due to their high sensitivity, simplicity, scope of scaling down for portability and low cost production [10]. Various enzyme- and molecular imprint-based amperometric biosensors for the detection of bilirubin have been reported [11], [12], [13], [14]. However, the enzyme-based methods have some inherent disadvantages, such as poor stability, high cost of the enzymes, and high sensitivity to environmental conditions [15]; moreover, the molecular imprint-based biomimetic sensors have yet to attain acceptable sensitivity in most cases [16]. Hence, the development of an alternative non-enzymatic biosensor with a suitable biorecognition system and proper selectivity for free bilirubin is warranted.

AuNCs typically consist of less than one hundred gold atoms, with sizes within the range of 0.3 to 3 nm. These nanoclusters are endowed with electronic characteristics that are significantly distinct from larger sized nanoparticles [17], [18]. The distinctive electronic nature of these nanoclusters is the origin of their different functional characteristics, among which the fluorescence properties are well documented [19]. The electrochemical properties of the AuNCs have yet to be adequately investigated, although the electrochemistry approach is well suited for the design and development of commercial biosensors. Some electrochemical and computational studies revealed that the redox properties of nanoclusters can be tuned effectively by controlling their core size [20]. Recently, redox active Au25 clusters have been reported in the electrochemical sensing of ascorbic acid, uric acid, dopamine [21] and glucose [22]. Moreover, nanoclusters conjugated with the redox mediator Azure A were reported to sense H2O2 non-enzymatically [23].

The stability of AuNCs is an important issue that must be resolved for their practical utility. The progress in creating AuNCs within protein matrices has stimulated interest in developing stable NCs [19], and we have recently implemented this concept to detect free bilirubin using an optical detection probe in a laboratory setting [24]. Notably, HSA is a natural carrier of bilirubin; however, the binding of bilirubin to this protein is not yet known to induce any significant decipherable signal that can be used to develop a detection method for free bilirubin. Here, we report that HSA-stabilized AuNCs are a biorecognition element that can be used to detect free bilirubin in an amperometric transducer-based biosensor platform. HSA-AuNCs were covalently immobilized onto Indium tin oxide (ITO) electrodes. The electrochemical investigation showed an interesting behavior of the HSA-AuNCs on the electrode surface for redox conversion of bilirubin, and the phenomena have been exploited for the sensitive detection of the free bilirubin levels in serum samples.

Section snippets

Materials

Bilirubin, Human serum albumin (HSA), Indium tin oxide-coated glass plates (ITO) (200–250 Ωcm 2), 3-Aminopropyltriethoxysilane (APTES), N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (EDC), N-Hydrosuccinimide (NHS) and auryl chloride (HAuCl4) were obtained from Sigma-Aldrich. All other reagents were of analytical reagent grade and used without further purification. Nanopure water (18.2 MΩ: Milipore Co., USA) was used throughout the experiment.

Synthesis, purification and characterization of the HSA-stabilized gold nanoclusters

We previously reported the synthesis and

Characterization of HSA-AuNC

The MALDI-MS spectra of the reaction mixture (Fig.2A) showed two peaks, 66.9 KDa and 70.5 KDa, corresponding to HSA and HSA-AuNC, respectively. After purification, only a single peak at 70.5 KDa was observed, confirming the successful purification of HSA-AuNC. The TEM analysis revealed well dispersed nanoclusters as dark spots in the TEM image, with average diameters of ~ 2.5 nm (Fig.2B and inset). The number of Au atoms present in an HSA protein matrix was 18 (Au18) as calculated using the

Conclusions

This is the first report of the use of HSA-AuNCs as a biorecognition element for the sensitive amperometric detection of unbound bilirubin in deproteinized serum samples. The dynamic range of the response covered the free bilirubin level that is normally present in hyperbilirubinemia conditions. The AuNCs in the protein matrix acted as an electronic bridge by contacting a specific redox active moiety of the HSA-attached bilirubin molecule and the polarized electrode. The HSA in the HSA-AuNCs

Acknowledgements

We acknowledge DBT, India grant no. BT/264/NE/NEITBP/2011, for financial assistance that enabled us to perform this work, and CIF, IITG for providing the TEM, EDX-SEM and AFM facilities.

Mallesh Santhosh received a M. Tech. degree in biotechnology from IIT Guwahati, India, in 2011. He is a doctoral student in the Department of Biosciences and Bioengineering, IIT Guwahati, India. His research interests include the development of enzyme/non-enzyme-based nano-biosensors for clinical diagnosis, understanding the protein–protein and protein–nanomaterial interactions, and studying electron transfer reactions of proteins and bioactive species.

References (50)

  • M.Ç. Canbaz et al.

    Fabrication of a highly sensitive disposable immunosensor based on indium tin oxide substrates for cancer biomarker detection

    Anal. Biochem.

    (2014)
  • I. Taurino et al.

    Superior sensing performance of multi-walled carbon nanotube-based electrodes to detect unconjugated bilirubin

    Thin Solid Films

    (2013)
  • I. Taurino et al.

    Efficient voltammetric discrimination of free bilirubin from uric acid and ascorbic acid by a CVD nanographite-based microelectrode

    Talanta

    (2014)
  • P.A. Zunszain et al.

    Crystallographic analysis of human serum albumin complexed with 4Z,15E-bilirubin-IX

    J. Mol. Biol.

    (2008)
  • P. Das et al.

    Highly sensitive and stable laccase based amperometric biosensor developed on nano-composite matrix for detecting pyrocatechol in environmental samples

    Sensors Actuators B Chem.

    (2014)
  • G. Cheng et al.

    In-situ UV–visible spectroelectrochemical and circular dichroic electrochemical study of bilirubin and bilirubin + human serum albumin complex

    Bioelectrochem. Bioenerg.

    (1994)
  • C. Slifstein et al.

    pH effects on the reduction of bilirubin in aqueous media

    J. Electroanal. Chem.

    (1977)
  • H.-B. Noh et al.

    Selective nonenzymatic bilirubin detection in blood samples using a Nafion/Mn–Cu sensor

    Biosens. Bioelectron.

    (2014)
  • L.C. Mireles et al.

    Antioxidant and cytotoxic effects of bilirubin on neonatal erythrocytes

    Pediatr. Res.

    (1999)
  • R.P. Wennberg

    The blood-brain barrier and bilirubin encephalopathy

    Cell. Mol. Neurobiol.

    (2000)
  • J.F. Watchko et al.

    Bilirubin-induced neurologic damage- mechanisms and management approaches

    N. Engl. J. Med.

    (2013)
  • B.T. Doumas et al.

    Candidate reference method for determination of total bilirubin in serum: development and validation

    Clin. Chem.

    (1985)
  • P. Bijster et al.

    On the standardisation of the direct spectrophotometric bilirubin determination. Influence of the albumin source and the molar bilirubin: albumin ratio

    Ann. Clin. Biochem.

    (1981)
  • C.E. Ahlfors et al.

    Unbound (free) bilirubin: improving the paradigm for evaluating neonatal jaundice

    Clin. Chem.

    (2009)
  • A.P.F. Turner

    Biosensors: sense and sensibility

    Chem. Soc. Rev.

    (2013)
  • Cited by (46)

    • Lysozyme protected copper nano-cluster: A photo-switch for the selective sensing of Fe<sup>2+</sup>

      2023, Journal of Photochemistry and Photobiology A: Chemistry
      Citation Excerpt :

      The physicochemical properties of the protein-MNC can be tuned by varying the metal composition, size of the metal core, type of capping ligand, nature and concentration of reducing agents, reaction conditions etc. [32,34,35]. Gold (Au)[7,11,14–16,36–43] and silver (Ag)[13,44–48] nanoclusters have extensively been explored among the various protein-protected noble metal nanoclusters. Due to their redox properties, Au and Ag were relatively easier to stabilize using the protein template [10].

    • Selective and sensitive fluorescence turn-on detection of bilirubin using resorcinol-sucrose derived carbon dot

      2022, Analytical Biochemistry
      Citation Excerpt :

      High-resolution scan of the 2p region of C showed two clear peaks at 952.3 and 933eV with an energy gap of 19.3 eV which indicated the presence of Cu in Cu2+. Apart from that, there are several charge transfer satellite peaks at 941, 945 and 961 eV (Fig. 5c), which confirms the presence of Cu2+ complexed to surface oxygen-containing functional groups on rsCDs [38,39]. Furthermore, the excited state FL lifetime of rsCD was reduced from 1.91 nS to 1.65 nS after the addition of Cu2+ which evidenced the formation of rsCD-Cu2+ complex.

    View all citing articles on Scopus

    Mallesh Santhosh received a M. Tech. degree in biotechnology from IIT Guwahati, India, in 2011. He is a doctoral student in the Department of Biosciences and Bioengineering, IIT Guwahati, India. His research interests include the development of enzyme/non-enzyme-based nano-biosensors for clinical diagnosis, understanding the protein–protein and protein–nanomaterial interactions, and studying electron transfer reactions of proteins and bioactive species.

    Somasekhar R. Chinnadayyala Dr.Chinnadayyala completed a M.Sc. degree in Biochemistry from Yogi Vemana University India, in 2008. Later, he completed a PhD degree in Biotechnology from IIT Guwahati, India, in 2015. Dr.Chinnadayyala has developed expertise in the field of nano-biosensors.

    Naveen K Singh received his B. Tech. degree in Biotechnology from Allahabad Agriculture Institute, India, in 2011 and M. E. Degree in Biotechnology from BITS PILANI, India, in 2013. He is a Ph.D. student in the Department of Biosciences and Bioengineering, IIT Guwahati, India. His research interest is the development of biosensors for the rapid detection of Malaria.

    Professor Pranab Goswami received his M.Sc. degree in chemistry from Gauhati University, India, in 1986 and subsequently received an MS degree in S & T, from BITS Pilani, Rajasthan, and a PhD degree from NEIST Jorhat (under Gauhati University) in 1994 in the area of biotechnology. From 1991 to 2002, he was a scientist (Scientist B to scientist E1) at NEIST, (CSIR) Jorhat, India. Prof. Goswami was a BOYSCAST fellow of DST, India, at University of Massachusetts Boston, USA, from 1996 to 97. He joined IIT Guwahati in 2002 as an Assistant Professor and subsequently became a Professor in 2009. Prof.Goswami was the founder and head of the Central Instrumental Facility at IIT Guwahati from 2004 to 2006, Head of the Biotechnology Department from 2006 to 2009, and Chairman of the IIT Guwahati Library Committee from 2012 to 2014. Currently, Prof.Goswami is the head of the Energy Center at IIT Guwahati. Prof.Goswami has > 20 years of experience in the field of biocatalysis, particularly in studies of enzymes and their utilization for various processes and in developing products. The current focus of Prof.Goswami's research is the utilization of various redox enzymes to develop biosensors and biofuel cells for clinical, biomedical and environmental applications. Prof.Goswami is also a member of the editorial board of Biocatalysis and Agricultural Biotechnology(Elsevier, ISSN: 1878-8181).

    View full text