Study of the stability of functionalized gold nanoparticles for the colorimetric detection of dipeptidyl peptidase IV

In this report, we investigated three stabilization strategies of gold nanoparticles and their practical application for the visual detection of dipeptidyl peptidase IV (DPP-IV). Citrate-capped gold nanoparticles (Au NPs) are generally unstable in high-ionic-strength samples. Au NPs are easily tagged with various proteins and biomolecules rich in amino acids, leading to important biomedical applications including targeted drug delivery, cellular imaging, and biosensing. The investigated assays were based on different modes of stabilization, such as the incorporation of polyethylene glycol (PEG) groups, stabilizer peptide, and bifunctionalization. Although all approaches provided highly stable Au NP platforms demonstrated by zeta potential measurements and resistance to aggregation in a high-ionic-strength saline solution, we found that the Au NPs modified with a separate stabilizer ligand provided the highest stability and was the only platform that demonstrated sensitivity to the addition of DPP-IV, whilst PEGylated and peptide-stabilized Au NPs showed no significant response

Chromogenic detection of dipeptidyl peptidase IV (DPP-IV) activity using peptide-functionalized gold nanoparticles

Metal nanoparticles offer a useful platform for a wide range of biological applications especially for biosensing, bioimaging and drug delivery. This thesis presents a body of original research describing the synthesis, characterisation and development of a novel and convenient biosensing assay for detection of dipeptidyl peptidase IV (DPP-IV) enzyme activity using peptide functionalized gold nanoparticles. The distinctive optical and physical properties of gold nanoparticles (Au NP) were harnessed for the development of a colorimetric assay for rapid sensing of DPP-IV activities and screening DPP-IV inhbitors. The citrate reduction method for Au NPs synthesis was optimised and several potential peptide substrates (GPDC, VP-EN-DC, C/G dipeptide, GPG-EN-PEG4-LA, GPDCALNNC) were designed to provide substrates that mimic the DPP-IV natural substrates. The performances of the substrate functionalized Au NPs were assessed for their appropriateness for the detection of the enzyme activity. Addition of DPP-IV to the solutions containing the functionalized Au NPs resulted in cleavage of the substrate and thus causing the aggregation of the Au NPs which in turn led to a shift of the surface plasmon peak toward longer wavelengths, and a change of the colour of the colloidal suspension from red to blue. Overall, real-time detection of DPP-IV activity over a broader range (0-40 U/L) with high selectivity and stability was obtained, thus providing a method that can be used to determine the levels of DPP-IV/CD26 in biological fluids such as serum and plasma. Further assay developments were conducted to overcome limitations encountered with the original Au NP assay, especially the narrow dynamic linear range and stability in high ionic strength solutions. Validation and comparison of the Au NP assay developed has revealed that this method is highly correlated to the gold standard chromogenic Gly-Pro-pNA method for detection of enzyme activity in biological samples. Very good recoveries (in the range 83.6 –114.9%) were obtained in spiked serum samples, which indicate that this assay could provide a suitable alternative for enzyme activity detection with the naked eye and without the need for sophisticated instruments. Investigations into the effects of incorporating different stabilizers in order to improve the stability of the peptide functionalized Au NP in high ionic strength solutions were also investigated. Gold nanoparticles have different shapes and structures and an alternative approach for detection of DPP-IV activity using gold nanorods due to their higher refractive index sensitivities was explored. As a conclusion, three out of five approaches, all utilising Au NP-ligand conjugates were demonstrated useful for the detection of the DPP-IV activity. The system developed here is portable and would permit on-site analysis of samples, which offers a real alternative approach from traditional assays and reduces the need for laboratory testing. The logical next step in this research would be the continuation of experiments to transform this test into a point of care testing device that could offer an early detection tool for disease management

Chromogenic detection of dipeptidyl peptidase IV (DPP-IV) activity using peptide-functionalized gold nanoparticles

Metal nanoparticles offer a useful platform for a wide range of biological applications especially for biosensing, bioimaging and drug delivery. This thesis presents a body of original research describing the synthesis, characterisation and development of a novel and convenient biosensing assay for detection of dipeptidyl peptidase IV (DPP-IV) enzyme activity using peptide functionalized gold nanoparticles. The distinctive optical and physical properties of gold nanoparticles (Au NP) were harnessed for the development of a colorimetric assay for rapid sensing of DPP-IV activities and screening DPP-IV inhbitors. The citrate reduction method for Au NPs synthesis was optimised and several potential peptide substrates (GPDC, VP-EN-DC, C/G dipeptide, GPG-EN-PEG4-LA, GPDCALNNC) were designed to provide substrates that mimic the DPP-IV natural substrates. The performances of the substrate functionalized Au NPs were assessed for their appropriateness for the detection of the enzyme activity. Addition of DPP-IV to the solutions containing the functionalized Au NPs resulted in cleavage of the substrate and thus causing the aggregation of the Au NPs which in turn led to a shift of the surface plasmon peak toward longer wavelengths, and a change of the colour of the colloidal suspension from red to blue. Overall, real-time detection of DPP-IV activity over a broader range (0-40 U/L) with high selectivity and stability was obtained, thus providing a method that can be used to determine the levels of DPP-IV/CD26 in biological fluids such as serum and plasma. Further assay developments were conducted to overcome limitations encountered with the original Au NP assay, especially the narrow dynamic linear range and stability in high ionic strength solutions. Validation and comparison of the Au NP assay developed has revealed that this method is highly correlated to the gold standard chromogenic Gly-Pro-pNA method for detection of enzyme activity in biological samples. Very good recoveries (in the range 83.6 –114.9%) were obtained in spiked serum samples, which indicate that this assay could provide a suitable alternative for enzyme activity detection with the naked eye and without the need for sophisticated instruments. Investigations into the effects of incorporating different stabilizers in order to improve the stability of the peptide functionalized Au NP in high ionic strength solutions were also investigated. Gold nanoparticles have different shapes and structures and an alternative approach for detection of DPP-IV activity using gold nanorods due to their higher refractive index sensitivities was explored. As a conclusion, three out of five approaches, all utilising Au NP-ligand conjugates were demonstrated useful for the detection of the DPP-IV activity. The system developed here is portable and would permit on-site analysis of samples, which offers a real alternative approach from traditional assays and reduces the need for laboratory testing. The logical next step in this research would be the continuation of experiments to transform this test into a point of care testing device that could offer an early detection tool for disease management

Optimization of gold nanoparticle-based real-time colorimetric assay of dipeptidyl peptidase IV activity

Dipeptidyl peptidase IV (DPP-IV also referred to as CD-26) is a serine protease enzyme with remarkable diagnostic and prognostic value in a variety of health and disease conditions. Herein, we describe a simple and real-time colorimetric assay for DPP-IV/CD-26 activity based on the aggregation of gold nanoparticles (AuNPs) functionalized with the peptide substrates: Gly-Pro-Asp-Cys (GPDC) or Val-Pro-ethylene diamine-Asp-Cys (VP-ED-DC). Cleavage of the substrates by DPP-IV resulted in aggregation of the AuNPs with accompanying color change in the solution from red to blue that was monitored using either a UV–visible spectrophotometer or by the naked eye. Factors, such as time course of the reaction, stability of the functionalized AuNPs and the structure of the substrate that influence the cleavage reaction in solution were investigated. The effects of potential interference from serum proteins (lysozyme, thrombin and trypsin) on the analytical response were negligible. The detection limits when GPDC or VP-EN-DC functionalized AuNPs were used for DPP-IV assay were 1.2 U/L and 1.5 U/L, respectively. The VP-EN-DC method was preferred for the quantitative determination of DPP-IV activity in serum because of its wide linear range 0–30 U/L compared to 0–12 U/L for the GPDC assay. Recoveries from serum samples spiked with DPP-IV activity, between 5 and 25 U/L, and using the VP-EN-DC modified AuNPs method ranged between 83.6% and 114.9%. The two colorimetric biosensors described here are superior to other conventional methods because of their simplicity, stability, selectivity and reliability

Gold nanoparticle-based colorimetric biosensors

Gold nanoparticles (AuNPs) provide excellent platforms for the development of colorimetric biosensors as they can be easily functionalised, displaying different colours depending on their size, shape and state of aggregation. In the last decade, a variety of biosensors have been developed to exploit the extent of colour changes as nano-particles (NPs) either aggregate or disperse, in the presence of analytes. Of critical importance to the design of these methods is that the behaviour of the systems has to be reproducible and predictable. Much has been accomplished in understanding the interactions between a variety of substrates and AuNPs, and how these interactions can be harnessed as colorimetric reporters in biosensors. However, despite these developments, only a few biosensors have been used in practice for the detection of analytes in biological samples. The transition from proof of concept to market biosensors requires extensive long-term reliability and shelf life testing, and modification of protocols and design features to make them safe and easy to use by the population at large. Developments in the next decade will see the adoption of user friendly biosensors for point-of-care and medical diagnosis as innovations are brought to improve the analytical performances and usability of the current designs. This review discusses the mechanisms, strategies, recent advances and perspectives for the use of AuNPs as colorimetric biosensors. Keywords: biosensors, colloids, gold nanoparticles, nanotechnology, surface plasmon resonance, enzymes, quantification

Development of HPLC Method for Simultaneous Determination of Ibuprofen and Chlorpheniramine Maleate

One of the most prevalent over-the-counter cold and cough medications is the chlorpheniramine maleate (CPM)–ibuprofen (IBF) combination. A reversed-phase high-performance liquid chromatography (RP-HPLC) method was effectively optimized and developed for the simultaneous detection of chlorpheniramine maleate and ibuprofen in a pharmaceutical formulation. The mobile phase for the RP-HPLC method was an isocratic combination of acetonitrile and 0.01 M acetate buffer at pH 3.8 (55:45; v/v) on an Eclipse Plus C18 reversed phase column. An ultraviolet (UV) detector with a wavelength of 225 nm was used to detect the analytes at a flow rate of 1.0 mL/min. CPM and IBF were satisfactorily eluted, with mean retention times of 2.09 and 6.27 min, respectively. The approach was shown to be linear (R2 > 0.9998 for CPM and 0.9992 for IBF), precise (% RSD 3.02% for CPM and 3.48% for IBF), accurate (% recoveries 97.7–98.9% for CPM and 101–104.5% for IBF), specific, easy to use, sensitive, quick, and robust. Limits of detection (LODs) were found to be 10 and 27 μg/mL for CPM and IBF, respectively. Without interference from excipients, the validated method could be utilized in regular quality control analysis of various dosage combinations of hard gelatin capsules containing CPM and IBF

Development of HPLC Method for Simultaneous Determination of Ibuprofen and Chlorpheniramine Maleate

One of the most prevalent over-the-counter cold and cough medications is the chlorpheniramine maleate (CPM)–ibuprofen (IBF) combination. A reversed-phase high-performance liquid chromatography (RP-HPLC) method was effectively optimized and developed for the simultaneous detection of chlorpheniramine maleate and ibuprofen in a pharmaceutical formulation. The mobile phase for the RP-HPLC method was an isocratic combination of acetonitrile and 0.01 M acetate buffer at pH 3.8 (55:45; v/v) on an Eclipse Plus C18 reversed phase column. An ultraviolet (UV) detector with a wavelength of 225 nm was used to detect the analytes at a flow rate of 1.0 mL/min. CPM and IBF were satisfactorily eluted, with mean retention times of 2.09 and 6.27 min, respectively. The approach was shown to be linear (R2 > 0.9998 for CPM and 0.9992 for IBF), precise (% RSD 3.02% for CPM and 3.48% for IBF), accurate (% recoveries 97.7–98.9% for CPM and 101–104.5% for IBF), specific, easy to use, sensitive, quick, and robust. Limits of detection (LODs) were found to be 10 and 27 μg/mL for CPM and IBF, respectively. Without interference from excipients, the validated method could be utilized in regular quality control analysis of various dosage combinations of hard gelatin capsules containing CPM and IBF

Applications of plasmonics : general discussion

a review of plasmonic effects in NANOPHOTONIC WAVE-GUIDE; FLUORESCENCE LIFETIME; METAL NANOPARTICLES; LIGHT; NANOSTRUCTURES; NANOPLASMONICS; SCATTERING; EMISSION; ANTENNAS; MIRRORS