EIS spectra of the HSA-AuNC modified ITO electrode in the presence (red dot) and absence (black dot) of bilirubin. Schematic representation of the structure of HSA (different colors indicate different subdomains) containing bound Au18 NCs and bound bilirubin (A).
Overview
- Disorder in bilirubin metabolism
- Importance of bilirubin detection
This compound is formed as a result of the breakdown of heme in the blood of mammals. In the second step, biliverdin is reduced to bilirubin by the catalytic activity of a cytosolic enzyme biliverdin reductase (Scheme 1.1) (Sticova and Jirsa, 2013; Tenhunen et al., 1969).
Current methods for detection of bilirubin in serum
- Optical based methods
- Direct spectroscopy
- Diazo based method
- Enzymatic oxidation based methods
- Spectro fluorometric based method
- Electrochemical based methods
- Enzymatic electrochemical detection of bilirubin
- Non-Enzymatic electrochemical detection of bilirubin
- Separation based techniques for bilirubin detection
- Non-invasive techniques for bilirubin detection
An enzyme-based fiber optic fluorescent biosensor was developed for the determination of bilirubin (Li et al., 1996). 17 al., (2011 and 2010) reported bilirubin-guided fluorescence quenching of tetracycline-europium (Eu3+) complex and oxytetracycline for bilirubin quantification.
Nanoparticle-protein conjugation: Application in developing biosensors
31 the size of the gap between the energy levels (Eδ) is the determining factor for AuNC fluorescence (Zheng et al., 2007). Xie and co-workers reported the first AuNCs stabilized by biomimetic protein synthesis (Xie et al., 2009).
Overview
HSA dissolves bilirubin through its binding and acts as a buffer that prevents the transfer of bilirubin from blood to tissue and thereby alleviates the toxic effect of bilirubin on the tissue. As a result, the interaction of HSA with bilirubin has attracted much attention (Blaha et al., 1997; Goncharova et al., 2013; Moosavi-Movahedi et al., 2007) and the phenomenon has been exploited to develop methods for determining free bilirubin in blood serum (Lamola et al., 1979). Studies have shown that the HSA acts as a chiral matrix that can selectively recognize the P form of the bilirubin enantiomer.
However, the binding of bilirubin to native HSA does not produce a strong signal for the analytical application of bilirubin detection phenomena.
Experimental approaches
- Reagents and stock solutions
- Synthesis of HSA-stabilized nanocluster
- Purification of HSA-stabilized nanocluster
- Spectroscopic characterization of HSA-stabilized nanocluster
- Circular dichroism and Fourier transform infrared spectroscopic study
- Matrix- assisted laser desorption ionization- mass spectroscopy and transmission
- Zeta potential study
- Interaction studies of HSA-AuNC with bilirubin through fluorescence and circular
The intact mass of HSA and HSA-AuNC was determined by performing matrix-assisted laser desorption ionization - mass spectrometry (MALDI-MS) (4800 plus MALDI TOF/TOF Analyzer, AB SCIEX, USA). Tryptophan fluorescence was monitored to study the interaction of HSA-AuNC and native HSA with bilirubin. A series of solutions containing a fixed concentration of HSA-AuNC (5 μM) and HSA (5 μM) with different concentrations of bilirubin (0–7 μM) were prepared in 50 mM PBS pH 7.4.
For CD measurement in the visible region, the concentrations of HSA and HSA-AuNC and bilirubin were set at 12 µM.
Results and discussion
- Spectroscopic characterization of HSA-AuNC
- Secondary structure of HSA-AuNC
- Isoelectric point (pI) of HSA-AuNC
- Interaction study of HSA-AuNC with bilirubin
- Fluorescence spectroscopic studies
- Far- UV CD spectroscopic studies
- UV-visible and Induced CD spectroscopic studies
CD measurements were performed with free HSA and HSA-AuNC systems to understand any secondary structure transitions of HSA after the formation of NCs in the protein. The fluorescence intensities of HSA solution and HSA-AuNC in the presence of bilirubin in solution were then studied. The effect of bilirubin binding on the secondary structure of HSA and HSA-AuNC was investigated through far-UV CD measurements.
It is interesting to note that both CD spectra of HSA and HSA-AuNC with bilirubin show bisignate features, indicating the formation of the.
Conclusions
For HSA-AuNC and bilirubin interaction, the CD spectrum resumes a similar bisignate form with a negative peak around 420 nm and a positive peak around 475 nm, indicating that HSA-AuNC binds to the P form of bilirubin. However, the red shift in the peak position with decrease in intensity implies a change in the binding site for bilirubin in HSA-AuNC. Inset: photographs under visible light (left) and ultraviolet light (right) of the HSA-AuNC solution.
Inset showing the percentage of secondary structure motifs of the proteins HSA and HSA-AuNC. B) FTIR spectra of HSA and HSA-AuNC.
- Overview
- Experimental Approaches
- Reagents and stock solutions
- Synthesis and purification of HSA-stabilized nanocluster
- Fluorescence-based detection of bilirubin
- Zeta-potential and time resolved fluorescence studies
- Catalytic activity of HSA-AuNC against bilirubin IX
- Kinetic studies of HSA-AuNC catalytic activity
- Preparation of human serum samples
- Results and discussion
- Fluorescence response of HSA-AuNC with bilirubin
- Catalytic activity of HSA-AuNC
- Kinetic parameters of HSA-AuNC towards bilirubin oxidation
- Detection of bilirubin in human serum
- Conclusions
The rate constant for oxidation of bilirubin by HSA-AuNC (Kp) was determined by measuring the decrease in A440nm (ΔA) after the addition of HSA-AuNC (10 μL) and H2O2 (5 μL) to dilute solutions of bilirubin (1, 2) and 3 µM) (volume made up to 1 ml with PBS buffer). The zeta potential of HSA-AuNC before and after addition of bilirubin was also measured. Zeta potential distribution of HSA-AuNC at pH 7.4, in the absence (B) and presence (C) of bilirubin.
Introduction of HSA-AuNC accelerated the electron transfer in the redox conversion of bilirubin at the electrode surface.
71
Overview
In the previous chapter, the concept of interaction of free bilirubin with its natural carrier HSA and the characteristic electronic nature of the nanoclusters was successfully implemented for the detection of bilirubin using HSA-AuNC as an optical detection probe in a laboratory setup. We would like to explore the electrochemical properties of HSA-AuNC here to understand the feasibility of using these nano-conjugates for quantitative detection of bilirubin. In particular, the electrochemical transducer-based platforms have been widely exploited to develop commercial biosensors due to their high sensitivity, simplicity, and the scope of reducing their sizes for portability and low-cost production (Ronkainen et al., 2010).
Electrochemical investigation revealed an interesting behavior of HSA-AuNC on the electrode surface for the redox conversion of bilirubin, and the phenomenon was exploited for the sensitive detection of free bilirubin in serum samples.
Experimental Approaches
- Reagents
- Synthesis and purification of HSA-stabilized nanocluster
- Preparation of HSA-AuNC modified ITO electrode
- Characterization of modified electrodes
- Docking studies
Ten microliters of the above solution was poured onto a silanized ITO plate and dried overnight at room temperature to allow covalent attachment of HSA-AuNC to the modified ITO plates (Scheme 4.1). For reproducibility studies, five different HSA-AuNC-modified ITO electrodes were prepared using this mentioned procedure. To study lifetime and reproducibility, the bioelectrode was washed with 0.1 M NaOH after each successive measurement.
The molecular visualization program PyMOL (DeLano, 2002) was used to view the 3D structures and measure the intermolecular distance between the nanocluster cores of the different models and bilirubin in the HSA-bound bilirubin structure.
Result and discussion
- Surface characterization of modified electrodes
- Electrochemical characterization of modified electrodes
- Response of HSA-AuNC/APTES/ITO electrodes to the detection of bilirubin
- Reproducibility, stability, interference and real sample analysis
The absence of a reducing peak in the spectra indicates the irreversible nature of the reactions. 78 produce any peak in the presence of bilirubin, indicating the isolating behavior of the HSA protein. For this study, 5 μM bilirubin was measured in the absence and presence of 5 μM of each of the potential interfering agents.
Storage The stability of the HSA-uNC-modified ITO electrode was assessed periodically at 1-day intervals for 7 days.
Conclusion
Overview
In this regard, paper was identified as a real choice (Li et al., 2012) due to the following advantages: (i) cheap, portable and easily available, (ii) compatible with. 99 external forces and (iv) can be easily modified to immobilize various biomolecules such as protein, DNA, small molecules, etc. (Kakoti et al., 2015). ZnO nanostructures are promising functional materials with characteristic properties such as, high surface area, non-toxic, biocompatible, chemically stable, showing biomimetic and high electron communication (Wang et al., 2006; Weintraub et al., 2010).
Furthermore, the high isoelectric point (pI ~ 9.5) of ZnO nanostructures makes the immobilization of biomolecules with low pI more practical (Zhao et al., 2009).
Experimental Approaches
- Reagents
- Fabrication of paper-based electrochemical device
- Preparation of conductive graphite ink
- Synthesis of ZnO nanorods over paper substrate
- Synthesis and immobilization of HSA-stabilized nanocluster
- Apparatus and measurements
- Detection procedure of the electrochemical device
ZnO nanorods were grown over the solidified initial ZnO layer by the solvothermal method (Manekkathodi et al., 2010). Purification of the synthesized HSA-AuNC was performed by the Zn2+ assisted precipitation method described in Section 2.2.3. Biocompatible conductive inks were characterized for resistance using a two-electrode data acquisition system (Agilent 34972A LXI).
Strip-1 containing graphite paste and in situ grown ZnO-NR over graphite served as control, while HSA-AuNC immobilized over in situ grown ZnO-NRs acted as bioelectrode for bilirubin detection.
Results and Discussion
- Characterizations of patterned paper and conductive ink
- Spectroscopic and thermal characterization of the ink
- Morphological characterization of µPED by FESEM
- Electrochemical characterization of the fabricated electrodes
- Parameters optimization
- Cyclic voltammetry
- Response characteristics of bioelectrode towards bilirubin
- Reproducibility and interference test
In the presence of bilirubin, the HSA-AuNC/ZnO-NR electrode assembly showed an increase in current density in forward and reverse CV scanning (Figure 5.6 blue trace). The nanocluster present in the HSA protein matrix induced the specific redox conversion of bilirubin. Saturation in peak current was observed beyond 35 μM bilirubin indicating saturation of HSA-AuNC binding sites with bilirubin above this concentration.
The relative standard deviation (RSD) value was found to be ~7.8%, indicating good repeatability of the electrode for the detection of bilirubin.
Conclusion
Mechanism of formation and conjugation of bilirubin
On the other hand, low serum bilirubin is associated with iron deficiency and coronary artery disease (Stocker et al., 1987). Bhutani et al. (2013) conducted a systematic review and meta-analysis to assess the national prevalence, mortality and kernicterus due to extreme hyperbilirubinemia (EHB) and Rhesus (Rh) sensitivity.
Scheme of diazo reaction involves in bilirubin detection
Due to the pH dependence of the enzyme for the oxidation of different fractions of bilirubin, it is unsuitable for routine laboratory measurements. An immobilized three-enzyme system containing glucose oxidase, BOx, and horseradish peroxidase (HRP) was also investigated for enhanced bilirubin oxidation (Daka et al., 1989).
Enzymatic oxidation of bilirubin by BOx and HRP
A) Preparation of AuNC within HSA protein matrix via microwave irradiation
Detection of free bilirubin by using HSA-AuNC as fluorometric probe (A) and
In this chapter, a paper-based microfluidic electrochemical device (µPED) was reported for the detection of bilirubin using HSA-AuNC as an electrochemical probe. The use of HSA-AuNC as an electrochemical probe for the sensitive amperometric detection of bilirubin is also reported here for the first time. Via zinc(II) protoporphyrin to the synthesis of poly(ZnPP-MAA-EGDMA) for imprinting and selective binding of bilirubin.
Ionic effect on the binding of bilirubin to the printed poly(methacrylic acid-co-ethylene glycol-dimethylacrylate). Human serum albumin stabilized gold nanoclusters as a novel fluorescent and colorimetric probe for the detection of bilirubin-IX.
Schematic representation on the covalent immobilization of HSA-AuNC to the
Reaction occurring at the bare ITO electrode in presence of bilirubin…
Meanwhile, the HSA-AuNC-immobilized ITO electrode showed a well-defined redox peak, with oxidation at + 0.3 V and reduction at + 0.25 V (versus Ag/AgCl), instead of the three consecutive oxidation peaks. However, the magnitude of the anodic peak current was higher than the cathodic peak current, indicating that the oxidation of bilirubin is more favorable than the reduction of the oxidized product attached to the bioelectrode. This led to higher electrocatalytic oxidation of the substrate on the bioelectrode, leading to an increase in peak height compared to the bare ITO electrode.
Thus, the immobilization of HSA-AuNC on silanized ITO increased the total electroactive surface area of the electrode, indicating that the 3-D nanocluster-embedded protein was layered on the electrode surface.
Reaction occurring at the HSA/ HSA-AuNC modified ITO electrode in presence
Thus, the orientation of the C1 carbon near the AuNCs could be the reason for the observed site-specific oxidation of bilirubin. In addition, the effect of pH on the potential response of the bioelectrode to bilirubin oxidation was studied. Response of HSA-AuNC/APTES/ITO electrodes to bilirubin detection. The bioelectrode response was examined with a new load of 66 ± The bioelectrode response was examined with a new load of 66 ± 7.4 µg cm-2 (0.94 nM) of HSA-AuNC on the electrode.
The operational stability of the electrode was investigated by subjecting a freshly prepared HSA-AuNC-modified ITO electrode to 5 µM bilirubin in PBS buffer (pH 7.4) and its response in 15 consecutive measurements over a period of 4 hours to assess.
Scheme of immobilization of HSA-AuNC over in-situ grown ZnO nanorods on
Synthesis of multi-walled carbon nanotubes-COOH/graphene/gold nanoparticles nanocomposite for simple determination of bilirubin in human blood serum. Investigation of the use of bilirubin oxidase to measure the apparent concentration of unconjugated bilirubin in human plasma. Determination of conjugated and total bilirubin in the serum of newborns, using bilirubin oxidase.
Resolution of ultrafast excited-state bilirubin kinetics in chloroform and binding to human serum albumin. Human serum albumin-stabilized gold nanocluster immobilized on in situ grown zinc oxide nanorods over a paper electrode for electrochemical detection of bilirubin. Mallesh Santhosh and Pranab Goswami* Dual Fluorometric/Colorimetric Assay Based on Human Serum Albumin Stabilized Gold Nanoclusters for Sensitive Detection of Bilirubin.
