A thesis that partially fulfilled the conditions for obtaining a diploma. In the next section, we tried to improve the analytical capabilities of the alkaline phosphatase biosensor.
Contents
Paper-based miniaturized immuno-sensor for naked-eye ALP detection based on digital image colorimetry integrated with smartphone. Clinically comparable impedometric determination of ALP based on electrochemically tuned Au-nano-dendroids-GO nanocomposite in human serum.
CV responses of the modified surfaces (i) SPCE (black), (ii) SPCE/AuNPs (red), (iii) SPCE/AuNPs/Au-nano-dendroid (blue), (iv) SPCE/AuNPs/Au- nano -dendroid /GO (gray), (v) SPCE/AuNPs/Au-nano-dendroid /GO/Anti-ALP (pink). Histogram obtained from shelf life study of the GCE/AuNPs/AuNRTs-rGO-Naf sensor probe tested with 10-4 M ST.
List of tables
Chapter- I
General introduction
The need of miniaturized diagnostics
Nevertheless, the nutritional values and quality of these processed/preserved foods have become a major concern (Amit et al., 2017). These regulatory bodies not only ensure quality production of processed food, but also check illicit production (Kotsanopoulos and Arvanitoyannis, 2017; Sharma et al., 2010).
Introduction to biomarkers: importance in diagnostics
Introduction to biosensors
- Classification of biosensors based on transducers
- Optical biosensor
- Mechanical biosensor
- Electrochemical biosensor
- Classification of biosensors based on bio-recognition elements
- Label and label-free biosensing strategies
- Label assisted biosensing methods
- Label-free biosensing methods
Electrochemical (EC) biosensors are another class of biosensors which use different electrochemical techniques (Prasad et al., 2016). When the target analyte itself is able to deliver the analytical signals, it is referred to as a label-free biosensor (Sang et al., 2016).
Biosensor probe development and characterization
UV-visible spectroscopy (UV-Vis) allows preliminary determination of the formation of particles or nanomaterials (Verma et al., 2014). During probe fabrication, these nanomaterials play an important role in the covalent immobilization of biorecognition elements (Baranwal et al., 2016).
Analytical performance of biosensor
- Linear dynamic range
- Limit of quantification (LOQ)
- Limit of detection (LOD)
- Selectivity
- Shelf-life
- Sensitivity
Where LOQ is the limit of quantification; SDBlank is the standard deviation of the blank and the slope indicates the sensitivity of the probe to a given analyte. Where LOD is the limit of detection, SDBlank is the standard deviation of the blank and the slope indicates the sensitivity of the probe to a given analyte.
Concept of surface engineering and importance of nanomaterials in biosensors
Another role of nanomaterials in biosensor fabrication is to provide stable immobilization of the biorecognition element to obtain higher sensitivity and amplified signal. These syntheses show the addition of atoms, followed by nucleation and then maturation of the structures (Mahato et al., 2019).
Support matrices for biosensors
- Paper as support matrices
- Carbon paste as support matrix
- Glassy carbon as support matrix
This section describes the importance of various support matrices, which are cheaper and widely used for low-cost biosensor developments. Not only that, paper matrices are also impregnated with various nanomaterials forming nano-paper electrodes, which have recently been used for electrochemical sensing of various molecules (Guo et al., 2018; Huang, 2018).
Techniques used for biosensors studies
- Techniques for sensor probe assessment
- Colorimetric assessment
- Electrochemical assessments of biosensor
- Techniques for real-sample analysis
- Spike and recovery method
- Standard addition method
Here, we observe an increase in resistance to surface charge transfer upon sequential addition of probe and target molecule components ( Rushworth et al., 2014 ). In this analysis, a sensor probe is used to obtain unknown concentrations of a target present in actual sample matrices.
Objectives and goals of the study
Objective #1: Paper-based miniaturized immunosensor for naked-eye ALP detection based on digital image colorimetry integrated with a smartphone. Objective #4: Novel electrochemical biosensor for serotonin detection based on gold nanoratchets decorated with reduced graphene oxide in biological fluids and in vitro model.
Thesis overview
Noh, H.-B., Chandra, P., Kim, Y.-J., Shim, Y.-B., A simple separation method with a microfluidic channel based on alternating current potential modulation, 2012. Zhu , Y. , Chandra, P. , Shim, Y.-B. , Ultrasensitive and selective electrochemical diagnosis of breast cancer based on a hydrazine–Au nanoparticle–aptamer bioconjugate, 2012b.
Chapter- II
Paper-based miniaturized immuno-sensor for naked eye ALP detection based on digital image
Introduction
In addition, it has also been reported that in the case of mastitis, the concentration of ALP in milk is extremely high (Babaei et al., 2007; Kitchen, 1981). This LFA-based method is based on the indirect detection of ALP using adsorbed antibodies in the sensor matrix (Yu et al., 2015). Stable immobilization of an antibody that preserves its biological activity can be achieved by conventional covalent immobilization procedures (Akhtar et al., 2018; Chandra et al., 2012).
Experimental
- Materials, apparatus, and reagent preparation
- Fabrication of P/DS/EDC-NHS/Anti-ALP biosensor
- Detection of ALP using the P/DS/EDC-NHS/Anti-ALP biosensor
The final sense probe after Anti-ALP immobilization was represented as P/DS/EDC-NHS/Anti-ALP probe. To minimize nonspecific interaction of ALP and avoid false positive signals, the P/DS/EDC-NHS/Anti-ALP probe was treated with 1%. ALP detection relies on the immunocomplexation reaction between the P/DS/EDC-NHS/Anti-ALP probe and ALP.
Schematic representation of the ALP biosensor fabrication and detection principle
- Results and discussion
- Selection of color channel for biosensing studies
- Characterization of P/DS/EDC-NHS/Anti-ALP biosensing probe
- Analytical performance of P/DS/EDC-NHS/Anti-ALP biosensor
- Interference study
- Real sample analysis
- Prototype design and ALP analysis in home kitchen
- Storage conditions, stability test, and reproducibility assay
- Conclusions
- References
The change in the color of the paper surface clearly shows the increase in Effective IRed. Where [A]ALP and [B]ALP are the analytical response of ALP in the supplement and blank, respectively; and [ ]C ALP is the analytical response of ALP in standard solutions. We also examined the shelf life of the probe, testing the Effective IRed P/DS/EDC-NHS/Anti-ALP probes for every third day.
Chapter- III
Clinically comparable impedimetric
Chemicals and instrumentation
The antibody of ALP (anti-ALP), graphite flakes (C), nafion (C7HF13O5S·C2F4) was procured from Sigma Aldrich, USA. Potassium Chloride (KCl), Potassium Permanganate (KMnO4), Potassium Ferrocynide (K4Fe(CN)6.3H2O), Potassium Ferricynide (K3Fe(CN)6), Sodium Chloride (NaCl), Monobasic Sodium Phosphate (NaH2PO4), Dibasic Sodium (Sodium Phosphate (Na2HPO4)) were purchased from SRL Research Laboratory, India. The acids solvents phosphoric acid (H3PO4), sulfuric acid (H2SO4), hydrochloric acid (HCl: 30%), ethanol (C2H5OH) and acetonitrile (C2H3N) were obtained from Merck Millipore.
Synthesis of graphene oxide
FTIR (Cary 630: Agilent, USA) and scanning electron microscope (SEM: Sigma: Carl Zeiss Ltd, Germany) were used to characterize the modified surfaces during the stepwise fabrication of the detection probe, where the FTIR spectra were recorded in the range between 400 and 4000 cm-1 wavenumbers. Ag/AgCl with a modulation power of 10 mV in the variable frequency range from 10 Hz to 1000 KHz. The analysis of the obtained spectra was carried out by obtaining the charge transfer resistance (Rct), which is parallel in the equivalent circuit of impedance spectra.
Digital image colorimetry based control study
Further DIC analysis in the control studies was also performed using the procedure followed by the technique reported earlier (Mahato and Chandra, 2019). In the next step, Au nano-dendroids were electrochemically synthesized on the SPCE/AuNPs modified surface. The final modified sensor probe was named as SPCE/AuNPs/Au-nano-dendroid/GO/anti-ALP.
Result and discussions
- Characterization of GO
- Analytical performance
- Selectivity assay
- Real sample studies
The fabrication of SPCE/AuNPs/Au-nano-dendroid/GO/anti-ALP sensor probe has been characterized using physical as well as electrochemical techniques. Finally, the fabricated sensing probe was designated as SPCE/AuNPs/Au-nano-dendroid/GO/anti-ALP. Au-nano-dendroid /GO/Anti-ALP modified sensor probe, (B) calibration plot obtained from the dose-dependent spectrum.
ALP (U/L)]
Storage conditions, stability, and reproducibility assessment
Kim, T.-I., Kim, H., Choi, Y., Kim, Y., A fluorescent light-on probe for detecting alkaline phosphatase activity in living cells, 2011. Lee, J.Y., Ahn, J.K., Park , K.S., Park, H.G., An impedimetric determination of alkaline phosphatase activity based on Cu2+-mediated oxidation reaction of DNA-bound poly-thymine, 2018. Wang, J.H., Wang, K., Bartling, B. , Liu, C. -C., Detection of alkaline phosphatase using an electrochemical biosensor in a one-step approach, 2009.
Chapter- IV
Development of miniaturized electrochemical nanobiosensor based on AuNPs-porous
Chemicals and instruments
All the chemicals used in the experiments were of AR/HPLC grade and were used without any purification step. All the electrochemical analyzes have been performed using electrochemical workstation (Autolab: Metrohm, The Netherlands) with the conventional three-cell electrode system comprising glassy carbon electrode (GCE) as working, platinum electrode as counter and Ag/AgCl electrode (in saturated KCl ) as reference electrode. The electrochemical impedance spectroscopy was performed using the frequency response analysis (FRA) module on the electrochemical workstation, where the spectra were recorded at open circuit potential (V) vs.
Synthesis of PG-DAN
To characterize the synthesized material, FTIR (Cary 630: Agilent, USA) and field emission electron microscope (FETEM: Jeol-2100F: Jeol Ltd. Japan), scanning electron microscope (SEM: Sigma: Carl Zeiss Ltd, Germany) ) were used. The resulting spectra were analyzed using a circuit consisting of a series-connected solution resistance and a parallel-connected charge transfer resistance (Rct). Chromatographic analyzes were performed using a Dionex ultimate 3000 UHPLC system synchronized with Chromoleon console version 7.2.8 software.
Sample preparation for high performance liquid chromatography
Fabrication of GCE/AuNPs/PG-DAN-Naf nanobiosensor probe
Diseased and healthy mice brain sample preparation
Mice were then sacrificed on day 7 by cervical dislocation and the brain was removed and dissected according to a standard protocol (Spijker, 2011). The excised homogenized brain was further used to prepare the sample for Vit-C analysis using the GCE/AuNPs/PG-DAN-Naf nanobiosensor probe.
Results and Discussions
- Characterization of the PG-DAN nanocomposite
- Characterization of GCE/AuNPs/PG-DAN-Naf nanobiosensor probe
- Analytical performance of GCE/AuNPs/PG-DAN-Naf nanobiosensor probe
- Vit-C detection in urine and fruit juice samples
- Vit-C detection in brain of healthy and diseased mice
Based on the results, the GCE/AuNPs/PG-DAN-Naf detection probe was used for analytical applications. GCE/AuNPs (image not shown), GCE/PG-DAN-Naf (image not shown), and GCE/AuNPs/PG-DAN-Naf (Figure 4.5). The GCE/AuNPs/PG-DAN-Naf nanobiosensor probe was further used for the detection of Vit-C.
Schematic representation for the process for (A) brain tissue isolation from mice (strain:C57bl/6j), (B) dissection of cortical tissues for the sample preparation
- Reproducibility and stability studies
The peak at 5.2 min was simply due to Vit-C, also confirmed by dose-dependent analysis under similar experimental conditions (left inset of Figure 4.12 B). After validating the standard Vit-C assay on HPLC, we tested Vit-C directly in healthy/diseased brain dialysate (figure 4.12 C-D). Interestingly, in this case significantly higher concentrations (p < 0.01) of Vit-C were observed in brain samples of healthy mice compared to diseased ones (inset figure 4.12 D).
Time (weeks)
Declaration
Li. , J., Krishnan, R.G., Nair, B.G., Satheesh Babu, T.G., Voltammetric determination of ascorbic acid using a disposable screen-printed electrode modified with Cu(OH)2 nanorods, 2017. Tang, Y., Wu , M., A rapid method for the simultaneous determination of ascorbic acid and sorbic acid in fruit juices by capillary zone electrophoresis, 2005.
Chapter- V
Novel electrochemical biosensor for serotonin detection based on gold nanorattles decorated
Synthesis of AuNRTs
Further, coating with a silver layer on it was achieved by treating a reducing solution of silver nitrate (0.5 mL: 10 mM) and ascorbic acid (2 mL: 0.1 M) onto the synthesized Au-octahedra, followed by thorough mixing. Ag-coated Au-octahedra were then extracted sequentially using PAH (6 mg/mL in 6 mM NaCl) and PVP (90 mM) solutions. In the last step for the synthesis of AuNRT, HAuCl4 was added dropwise to Ag-coated Au-octahedrons at moderate boiling.
AuNRTs-rGO-Naf nanocomposite preparation and fabrication of GCE/AuNPs/AuNRTs-rGO-Naf sensor probe
Preparation of AuNRTs-rGO-Naf nanocomposite and fabrication of GCE/AuNPs/AuNRTs-rGO-Naf sensor probe:.
Mammalian cell culture and human serum sample preparation
Characterization of the AuNRTs-rGO nanocomposite
A dose-dependent plot was obtained using the UV-Vis responses, showing linear increase of peaks at λmax (657 nm) indicating that the peak λmax is simply due to the AuNRTs (inset figure 5.1B). In the UV-Vis spectrum corresponding to rGO (figure 5.1 A (ii); red), an absorption peak near λmax. 260 nm) was observed due to the optical absorption of π-π* transition of aromatic C=C, C=O and C–O bonds. The representative HR-TEM micrograph and SAED pattern are shown in figure 5.3(A) and 5.3(B) respectively.
Characterization of GCE/AuNPs/AuNRTs-rGO-Naf sensor probe
Therefore, for the further analyses, we have used GCE/AuNPs/AuNRTs-rGO-Naf as a sensor probe. It is interesting to note that the lowest Rct was obtained for GCE/AuNPs/AuNRTs-rGO-Naf surface compared to other tested surfaces. GCE/AuNPs/AuNRTs-Naf, GCE/AuNPs/AuNRTs-rGO-Naf modified electrode surfaces, (B) histogram showing the respective Rct values obtained from the EIS spectra.
Analytical performance of the GCE/AuNPs/AuNRT-rGO-Naf sensor probe
PBS containing 10-3 M ST using bare GCE probe where a broad peak at 0.39 V was observed, which is due to the presence of ST in electrolyte solution. The signal response of the GCE/AuNPs/AuNRTs-rGO-Naf sensor surface was found with the two-fold increase than the bare GCE in the presence at the same concentration of ST (i.e. 10-3M), indicating that the fabricated biosensor is capable to sensitive determination of ST (Figure 5.8 (A). To validate the peak observed at 0.37 V was simply due to the interaction of ST with the sensor probe, we performed two separate control experiments.
Selectivity assay
Where, [ ]S ST and [ ]B ST are the analytical responses of ST in spiked and blank urine samples, respectively; and [SS]ST is the analytical response of ST in standard solutions. Further, the fabricated probe was challenged for the determination of ST in relatively complex matrices. For this purpose, the fabricated probe was used for the determination of ST in clinical serum samples.
Log conc. [ST (M)]
