The identification of rice bacterial leaf blight disease requires a simple, rapid, highly sensitive, and quantitative approach that can be applied as an early detection monitoring tool in rice health. This paper highlights the development of a turn-off fluorescence-based immunoassay for the early detection of Xanthomonas oryzae pv. oryzae (Xoo), a gram-negative bacterium that causes rice bacterial leaf blight disease. Antibodies against Xoo bacterial cells were produced as specific bio-recognition molecules and the conjugation of these antibodies with graphene quantum dots and gold nanoparticles was performed and characterized, respectively. The combination of both these bio-probes as a fluorescent donor and metal quencher led to changes in the fluorescence signal. The immunoreaction between AntiXoo-GQDs, Xoo cells, and AntiXoo-AuNPs in the immuno-aggregation complex led to the energy transfer in the turn-off fluorescence-based quenching system. The change in fluorescence intensity was proportional to the logarithm of Xoo cells in the range of 100-105 CFU mL-1. The limit of detection was achieved at 22 CFU mL-1 and the specificity test against other plant disease pathogens showed high specificity towards Xoo. The detection of Xoo in real plant samples was also performed in this study and demonstrated satisfactory results.
In the present research, we report a greener, faster, and low-cost synthesis of gold-coated iron oxide nanoparticles (Fe3 O4 /Au-NPs) by different ratios (1:1, 2:1, and 3:1 molar ratio) of iron oxide and gold with natural honey (0.5% w/v) under hydrothermal conditions for 20 minutes. Honey was used as the reducing and stabilizing agent, respectively. The nanoparticles were characterized by X-ray diffraction (XRD), UV-visible spectroscopy, field emission scanning electron microscope (FESEM), energy-dispersive X-ray spectroscopy (EDXS), transmission electron microscopy (TEM), selected area electron diffraction (SAED), vibrating sample magnetometer (VSM), and fourier transformed infrared spectroscopy (FT-IR). The XRD analysis indicated the presence of Fe3 O4 /Au-NPs, while the TEM images showed the formation of Fe3 O4 /Au-NPs with diameter range between 3.49 nm and 4.11 nm. The VSM study demonstrated that the magnetic properties were decreased in the Fe3 O4 /Au-NPs compared with the Fe3 O4 -NPs. The cytotoxicity threshold of Fe3 O4 /Au-NPs in the WEHI164 cells was determined by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. It was demonstrated no significant toxicity in higher concentration up to 140.0 ppm which can become the main candidates for biological and biomedical applications, such as drug delivery.
In this paper, the electrochemical behavior of myricetin on a gold nanoparticle/ethylenediamine/multi-walled carbon-nanotube modified glassy carbon electrode (AuNPs/en/MWCNTs/GCE) has been investigated. Myricetin effectively accumulated on the AuNPs/en/MWCNTs/GCE and caused a pair of irreversible redox peaks at around 0.408 V and 0.191 V (vs. Ag/AgCl) in 0.1 mol L-1 phosphate buffer solution (pH 3.5) for oxidation and reduction reactions respectively. The heights of the redox peaks were significantly higher on AuNPs/en/MWNTs/GCE compare with MWCNTs/GC and there was no peak on bare GC. The electron-transfer reaction for myricetin on the surface of electrochemical sensor was controlled by adsorption. Some parameters including pH, accumulation potential, accumulation time and scan rate have been optimized. Under the optimum conditions, anodic peak current was proportional to myricetin concentration in the dynamic range of 5.0×10-8 to 4.0×10-5 mol L-1 with the detection limit of 1.2×10-8 mol L-1. The proposed method was successfully used for the determination of myricetin content in tea and fruit juices.
In this study, an electrochemical sensor was fabricated based on gold nanoparticles/ ethylenediamine/ multi-wall carbon-nanotubes modified gold electrode (AuNPs/en/MWCNTs/AuE) for determination of valrubicin in biological samples. Valrubicin was effectively accumulated on the surface of AuNPs/en/MWCNTs/AuE and produced a pair of redox peaks at around 0.662 and 0.578V (vs. Ag/AgCl) in citrate buffer (pH4.0). The electrochemical parameters including pH, buffer, ionic strength, scan rate and size of AuNPs have been optimized. There was a good linear correlation between cathodic peak current and concentration of valrubicin in the range of 0.5 to 80.0μmolL(-1) with the detection limit of 0.018μmolL(-1) in citrate buffer (pH4.0) and 0.1molL(-1) KCl. Finally, the constructed sensor was successfully applied for determination of valrubicin in human urine and blood serum. In further studies, the different sequences of single stranded DNA probes have been immobilized on the surface of AuNPs decorated on MWCNTs to study the interaction of oligonucleotides with valrubicin.
Highly oriented ZnO nanorod (NR) arrays were fabricated on a seeded substrate through a hydrothermal route. The prepared ZnO nanorods were used as an amperometric enzyme electrode, in which glucose oxidase (GOx) was immobilised through physical adsorption. The modified electrode was designated as Nafion/GOx/ZnO NRs/ITO. The morphology and structural properties of the fabricated ZnO nanorods were analysed using field-emission scanning electron microscope and X-ray diffractometer. The electrochemical properties of the fabricated biosensor were studied by cyclic voltammetry and amperometry. Electrolyte pH, electrolyte temperature and enzyme concentration used for immobilisation were the examined parameters influencing enzyme activity and biosensor performance. The immobilised enzyme electrode showed good GOx retention activity. The amount of electroactive GOx was 7.82 × 10-8 mol/cm2, which was relatively higher than previously reported values. The Nafion/GOx/ZnO NRs/ITO electrode also displayed a linear response to glucose ranging from 0.05 mM to 1 mM, with a sensitivity of 48.75 µA/mM and a low Michaelis-Menten constant of 0.34 mM. Thus, the modified electrode can be used as a highly sensitive third-generation glucose biosensor with high resistance against interfering species, such as ascorbic acid, uric acid and L-cysteine. The applicability of the modified electrode was tested using human blood samples. Results were comparable with those obtained using a standard glucometer, indicating the excellent performance of the modified electrode.
This work presents a simple green synthesis of gold nanoparticles (AuNPs) by using an aqueous extract of Etlingera elatior (torch ginger). The metabolites present in E. elatior, including sugars, proteins, polyphenols, and flavonoids, were known to play important roles in reducing metal ions and supporting the subsequent stability of nanoparticles. The present work aimed to investigate the ability of the E. elatior extract to synthesise AuNPs via the reduction of gold (III) chloride hydrate and characterise the properties of the nanoparticles produced. The antioxidant properties of the E. elatior extract were evaluated by analysing the total phenolic and total flavonoid contents. To ascertain the formation of AuNPs, the synthesised particles were characterised using the ultraviolet-visible (UV-Vis) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, high-resolution transmission electron microscopy (HRTEM), energy-dispersive X-ray (EDX) microscopy, and dynamic light scattering (DLS) measurement. The properties of the green synthesised AuNPs were shown to be comparable to the AuNPs produced using a conventional reducing agent, sodium citrate. The UV-Vis measured the surface plasmon resonance of the AuNPs, and a band centered at 529 nm was obtained. The FTIR results proved that the extract contained the O-H functional group that is responsible for capping the nanoparticles. The HRTEM images showed that the green synthesized AuNPs were of various shapes and the average of the nanoparticles' hydrodynamic diameter was 31.5 ± 0.5 nm. Meanwhile, the zeta potential of -32.0 ± 0.4 mV indicates the high stability and negative charge of the AuNPs. We further successfully demonstrated that using the green synthesised AuNPs as the nanocomposite to modify the working surface of screen-printed carbon electrode (SPCE/Cs/AuNPs) enhanced the rate of electron transfer and provided a sensitive platform for the detection of Cu(II) ions.
Deoxyribonucleic acid or DNA molecules expressed as double-stranded (DSS) negatively charged polymer plays a significant role in electronic states of metal/silicon semiconductor structures. Electrical parameters of an Au/DNA/ITO device prepared using self-assembly method was studied by using current-voltage (I-V) characteristic measurements under alpha bombardment at room temperature. The results were analyzed using conventional thermionic emission model, Cheung and Cheung's method and Norde's technique to estimate the barrier height, ideality factor, series resistance and Richardson constant of the Au/DNA/ITO structure. Besides demonstrating a strongly rectifying (diode) characteristic, it was also observed that orderly fluctuations occur in various electrical parameters of the Schottky structure. Increasing alpha radiation effectively influences the series resistance, while the barrier height, ideality factor and interface state density parameters respond linearly. Barrier height determined from I-V measurements were calculated at 0.7284 eV for non-radiated, increasing to about 0.7883 eV in 0.036 Gy showing an increase for all doses. We also demonstrate the hypersensitivity phenomena effect by studying the relationship between the series resistance for the three methods, the ideality factor and low-dose radiation. Based on the results, sensitive alpha particle detectors can be realized using Au/DNA/ITO Schottky junction sensor.
C-reactive protein (CRP) is an acute phase reactant to be a marker of inflammation and has been correlated with the cardiac injury. An immunoassay was performed using anti-human CRP antibody on an InterDigitated electrode (IDE) sensor to determine and specify CRP concentration for diagnosing the condition of myocardial inflammation. To promote the detection, gold nanoparticle (GNP) was seeded on the aminated-IDE surface. Anti-CRP was hitched on the GNP-seeded surface and identified the abundance of CRP. The limit of quantification was found as 100 fM, and the higher current response was noticed by increasing CRP concentrations with the sensitivity at 1 pM. Furthermore, CRP-spiked human serum did not interfere the determination of CRP and increased the current response, indicating suitability for a real-life sample. Similarly, the control experiments with nonimmune antibody Troponin I are not showing the definite current responses, proving the selective identification of CRP. This method of diagnosing is needful to determine the cardiovascular injury at the right time.
Vibrio cholerae is a Gram-negative bacterium that causes cholera, a diarrheal disease. Cholera is widespread in poor, under-developed or disaster-hit countries that have poor water sanitation. Hence, a rapid detection method for V. cholerae in the field under these resource-limited settings is required. In this paper, we describe the development of an electrochemical genosensor assay using lyophilized gold nanoparticles/latex microsphere (AuNPs-PSA) reporter label. The reporter label mixture was prepared by lyophilization of AuNPs-PSA-avidin conjugate with different types of stabilizers. The best stabilizer was 5% sorbitol, which was able to preserve the dried conjugate for up to 30 days. Three methods of DNA hybridization were compared and the one-step sandwich hybridization method was chosen as it was fastest and highly specific. The performance of the assay using the lyophilized reagents was comparable to the wet form for detection of 1aM to 1fM of linear target DNA. The assay was highly specific for V. cholerae, with a detection limit of 1fM of PCR products. The ability of the sensor is to detect LAMP products as low as 50ngµl(-1). The novel lyophilized AuNPs-PSA-avidin reporter label with electrochemical genosensor detection could facilitate the rapid on-site detection of V. cholerae.
An electrochemical aptasensor is described for determination of the phytohormone of zearalenone (ZEA). The gold electrode was modified with ZEA via covalent attachment using cysteamine-hydrochloride and 1,4-phenylene diisocyanate linker. A truncated ZEA aptamer with a dissociation constant of 13.4 ± 2.1 nM was used in an aptasensor. The electrochemical property was investigated using square wave voltammetry for monitoring the change in the electron transfer using the ferro/ferricyanide system as redox probe. Under optimal experimental conditions, the response was best measured at a potential of 0.20 V (vs. Ag/AgCl). The signals depended on the competitive mechanism between the immobilised ZEA and free ZEA for the aptamer binding site. The aptasensor works in the range 0.01 to 1000 ng·mL-1 ZEA concentration, with a detection limit of 0.017 ng·mL-1. High degree of cross-reactivity with the other analogues of ZEA was observed, whereas none towards other mycotoxins. The aptasensor was further applied for the determination of ZEA in the extract of maize grain and showed good recovery percentages between 87 and 110%. Graphical abstract Schematic representation of the electrochemical determination of zearalenone based on indirect competitive assay. Step a Immobilisation of ZEA on the surface of gold electrode via covalent attachment, b competition for the ZEA aptamer binding site between immobilised and free ZEA, and c current signal of the binding event based on SWV technique.
In this work, we proposed a biosensor for trypsin proteolytic activity assay using immobilization of model peptides on screen-printed electrodes (SPE) modified with gold nanoparticles (AuNPs) prepared by electrosynthetic method. Sensing of proteolytic activity was based on electrochemical oxidation of tyrosine residues of peptides. We designed peptides containing N-terminal cysteine residue for immobilization on an SPE, modified with gold nanoparticles, trypsin-specific cleavage site and tyrosine residue as a redox label. The peptides were immobilized on SPE by formation of chemical bonds between mercapto groups of the N-terminal cysteine residues and AuNPs. After the incubation with trypsin, time-dependent cleavage of the immobilized peptides was observed by decline in tyrosine electrochemical oxidation signal. The kinetic parameters of trypsin, such as the catalytic constant (kcat), the Michaelis constant (KM) and the catalytic efficiency (kcat/KM), toward the CGGGRYR peptide were determined as 0.33 ± 0.01 min-1, 198 ± 24 nM and 0.0016 min-1 nM-1, respectively. Using the developed biosensor, we demonstrated the possibility of analysis of trypsin specificity toward the peptides with amino acid residues disrupting proteolysis. Further, we designed the peptides with proline or glutamic acid residues after the cleavage site (CGGRPYR and CGGREYR), and trypsin had reduced activity toward both of them according to the existing knowledge of the enzyme specificity. The developed biosensor system allows one to perform a comparative analysis of the protease steady-state kinetic parameters and specificity toward model peptides with different amino acid sequences.
Bacterial and fungal infections are challenging due to their low susceptibility and resistance to antimicrobial drugs. For this reason, antimicrobial peptides (AMP) emerge as excellent alternatives to overcome these problems. At the same time, their active insertion into the cell wall of microorganisms can be availed for biorecognition applications in biosensing platforms. Temporin-PTA (T-PTA) is an AMP found in the skin secretions of the Malaysian fire frog Hylarana picturata, which presents antibacterial activity against MRSA, Escherichia coli, and Bacillus subtilis. In this work, T-PTA was explored as an innovative sensing layer aiming for the electrochemical differentiation of Klebsiella pneumoniae, Acinetobacter baumannii, Bacillus subtilis, Enterococcus faecalis, Candida albicans, and C. tropicalis based on the structural differences of their membranes. The biosensor was analyzed through electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). In this approach, the different structural features of each microorganism resulted in different adherence degrees and, therefore, different electrochemical responses. The transducing layer was fabricated by the self-assembling of a 4-mercaptobenzoic acid (MBA) monolayer and gold-capped magnetic nanoparticles (Fe3O4@Au) implemented to improve the electrical signal of the biointeraction. We found that each interaction, expressed in variations of electron transfer resistance and anodic peak current, demonstrated a singular response from which the platform can discriminate all different microorganisms. We found expressive sensitivity towards Gram-negative species, especially K. pneumoniae. A detection limit of 101 CFU.mL-1 and a linear range of 101 to 105 CFU.mL-1 were obtained. The T-PTA biosensor platform is a promising and effective tool for microbial identification.
A new electrochemical biosensor is described for voltammetric detection of gene sequence related to Trichoderma harzianum. The sensor involves immobilization of a 20 base single-stranded probe (ssDNA), which is complementary to a specific gene sequence related to T. harzianum on a gold electrode through specific adsorption. The DNA probe was used to determine the amount of target gene in solution using methylene blue (MB) as the electrochemical indicator. The covalently immobilized probe could selectively hybridize with the target DNA to form a hybrid on the surface despite the bases being attached to the electrode. The changes in the peak currents of methylene blue (MB), an electroactive label, were observed upon hybridization of probe with the target. Peak currents were found to increase in the following order: hybrid-modified AuE and the probe-modified AuE which localized to the affinity of MB. Control experiments with the non-complementary oligonucleotides were performed to assess whether the DNA biosensor responds selectively, via hybridization, to the target. DNA biosensor also able to detect microorganism at the species levels without nucleic acid amplification. The redox current was linearly related to the concentration of target oligonucleotide DNA, ranged from 1-20 ppm. Numerous factors, affecting the probe immobilization, target hybridization and indicator binding reactions are optimized to maximize the sensitivity and reduce the assay time.
This work presents an experimental study of gold-DNA-gold structures in the presence and absence of external magnetic fields with strengths less than 1,200.00 mT. The DNA strands, extracted by standard method were used to fabricate a Metal-DNA-Metal (MDM) structure. Its electric behavior when subjected to a magnetic field was studied through its current-voltage (I-V) curve. Acquisition of the I-V curve demonstrated that DNA as a semiconductor exhibits diode behavior in the MDM structure. The current versus magnetic field strength followed a decreasing trend because of a diminished mobility in the presence of a low magnetic field. This made clear that an externally imposed magnetic field would boost resistance of the MDM structure up to 1,000.00 mT and for higher magnetic field strengths we can observe an increase in potential barrier in MDM junction. The magnetic sensitivity indicates the promise of using MDM structures as potential magnetic sensors.
Exploring the new catalytic systems for the reduction of organic and inorganic pollutants from an indispensable process in chemical, petrochemical, pharmaceutical and food industries, etc. Hence, in the present work, authors motivated to synthesize bare reduced graphene oxide (rGO), polyaniline (PANI), three different ratios of rGO-PANI(80:20,50:50, 10:90) composites and rGO-PANI(80:20,50:50, 10:90) supported mono (Pd) & bimetallic [Pd: Au(1:1,1:2, 2:1)] nanocomposite by a facile chemical reduction method. Also, it investigated their catalytic performances for the reduction of organic/inorganic pollutants and antimicrobial activities. All the freshly prepared bare rGO, PANI, three different ratios of rGO-PANI(80:20, 50:50,10:90) composites and rGO-PANI(80:20, 50:50,10:90)/Pd & Pd: Au(1:1, 1:2,2:1) nanocomposite hybrid catalysts were characterized using UV-Vis, FT-IR, SEM, FE-SEM, EDAX, HR-TEM, XRD, XPS and Raman spectroscopy analysis. Among them, an optimized best composition of rGO-PANI(80:20)/Pd: Au(1:1) bimetallic nanocomposite hybrid catalyst exhibits better catalytic reduction and antimicrobial activities than other composites, as a result of strong electrostatic interactions between rGO, PANI and bimetal (Pd: Au) NPs through a synergistic effect. Hence, an optimized rGO-PANI(80:20)/Pd:Au(1:1) bimetallic nanocomposite catalyst would be considered as a suitable catalyst for the reduction of different nitroarenes, organic dyes, heavy metal ions and also significantly inhibit the growth of S. aureus, S. Typhi as well as Candida albicans and Candida kruesi in wastewater.
Surface enhanced Raman scattering (SERS) DNA biosensing is an ultrasensitive, selective, and rapid detection technique with the ability to produce molecule-specific distinct fingerprint spectra. It supersedes the long amplicon based PCR assays, the fluorescence and spectroscopic techniques with their quenching and narrow spectral bandwidth, and the electrochemical detection techniques using multiplexing. However, the performance of the SERS DNA biosensor relies on the DNA probe length, platform composition, both the presence and position of Raman tags and the chosen sensing strategy. In this context, we herein report a SERS biosensor based on dual nanoplatforms with a uniquely designed Raman tag (ATTO Rho6G) intercalated short-length DNA probe for the sensitive detection of the pig species Sus scrofa. In the design of the signal probe (SP), a Raman tag was incorporated adjacent to the spacer arm, followed by a terminal thiol modifier, which consequently had a strong influence on the SERS signal enhancement. The detection strategy involves the probe-target DNA hybridization mediated coupling of the two platforms, i.e., the graphene oxide-gold nanorod (GO-AuNR) functionalized capture probe (CP) and SP-conjugated gold nanoparticles (AuNPs), consequently enhancing the SERS intensity by both the electromagnetic hot spots generated at the junctions or interstices of the two platforms and the chemical enhancement between the AuNPs and the adsorbed intercalated Raman tag. This dual platform based SERS DNA biosensor exhibited outstanding sensitivity in detecting pork DNA with a limit of detection (LOD) of 100 aM validated with DNA extracted from a pork sample (LOD 1 fM). Moreover, the fabricated SERS biosensor showed outstanding selectivity and specificity for differentiating the DNA sequences of six closely related non-target species from the target DNA sequences with single and three nucleotide base-mismatches. Therefore, the developed short-length DNA linked dual platform based SERS biosensor could replace the less sensitive traditional methods of pork DNA detection and be adopted as a universal detection approach for the qualitative and quantitative detection of DNA from any source.
Cholera is a communicable disease caused by consumption of contaminated food and water. This potentially fatal intestinal infection is characterised by profuse secretion of rice watery stool that can rapidly lead to severe dehydration and shock, thus requiring treatment to be given immediately. Epidemic and pandemic cholera are exclusively associated with Vibrio cholerae serogroups O1 and O139. In light of the need for rapid diagnosis of cholera and to prevent spread of outbreaks, we have developed and evaluated a direct one-step lateral flow biosensor for the simultaneous detection of both V. cholerae O1 and O139 serogroups using alkaline peptone water culture. Serogroup specific monoclonal antibodies raised against lipopolysaccharides (LPS) were used to functionalize the colloidal gold nanoparticles for dual detection in the biosensor. The assay is based on immunochromatographic principle where antigen-antibody reaction would result in the accumulation of gold nanoparticles and thus, the appearance of a red line on the strip. The dry-reagent dipstick format of the biosensor ensure user-friendly application, rapid result that can be read with the naked eyes and cold-chain free storage that is well-suited to be performed at resource-limited settings.
In the present work, nanohybrid of an anticancer drug, doxorubicin (Dox) loaded gold-coated superparamagnetic iron oxide nanoparticles (SPIONs@Au) were prepared for a combination therapy of cancer by means of both hyperthermia and drug delivery. The Dox molecules were conjugated to SPIONs@Au nanoparticles with the help of cysteamine (Cyst) as a non-covalent space linker and the Dox loading efficiency was investigated to be as high as 0.32 mg/mg. Thus synthesized particles were characterized by HRTEM, UV-Vis, FT-IR, SQUID magnetic studies and further tested for heat and drug release at low frequency oscillatory magnetic fields. The hyperthermia studies investigated to be strongly influenced by the applied frequency and the solvents used. The Dox delivery studies indicated that the drug release efficacy is strongly improved by maintaining the acidic pH conditions and the oscillatory magnetic fields, i.e. an enhancement in the Dox release was observed from the oscillation of particles due to the applied frequency, and is not effected by heating of the solution. Finally, the in vitro cell viability and proliferation studies were conducted using two different immortalized cell lines containing a cancerous (MCF-7 breast cancer) and non-cancerous H9c2 cardiac cell type.
A dinuclear gold(I) pyrrolidinedithiocarbamato complex (1) with a bidentate carbene ligand has been constructed and shows potent in vitro cytotoxic activities towards cisplatin-resistant ovarian cancer cells A2780cis. Its rigid scaffold enables a zinc(II)-based metal-organic framework (Zn-MOF) to be used as a carrier in facilitating the uptake and release of 1 in solutions. Instead of using a conventional dialysis approach for the drug-release testing, in this study, a set of transwell assay-based experiments have been designed and employed to examine the cytotoxic and antimigratory activities of 1@Zn-MOF towards A2780cis.
Salmonella and Shigella genera are common pathogens that contaminate foods and beverages. Lateral flow assays (LFA) are commonly used to detect these pathogens. However, most of the developed LFAs are for single detection. Simultaneous detection of pathogens is required to reduce cost and time. In this work, 40 nm gold nanoparticles (AuNPs) were synthesized using the seeding growth method as labeling agent. The AuNPs were characterized and conjugated with mouse anti-Gram negative endotoxin antibody. The nitrocellulose membrane HF135 was immobilized with anti-mouse IgG antibody as a control line and two separate test lines with either anti-Shigella or anti-Salmonella antibody, respectively. Color intensity of test lines was observed for positive samples. A milk sample was used as proof of concept to mimic actual contamination. The limit of detection of the LFA was 3.0 × 106 CFU/mL for multiplex detection of Shigella flexneri and Salmonella Typhi and for both single detections. The result was comparable with the enzyme-linked immunosorbent assay (ELISA) analysis. The produced LFA could differentiate between Shigella flexneri, Shigella boydii, Salmonella Enteritidis, and Salmonella Typhi. The developed LFA was able to identify Shigella flexneri and Salmonella Typhi with good sensitivity in milk samples, thus, beneficial to ensure the safety of food before entering the market.