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