Graphene decorated with graphitic nanospheres functionalized with pyrene butyric acid (PBA) is used for the first time to fabricate a DNA biosensor. The electrode was formed by attaching a DNA probe onto PBA, which had been stacked onto a graphene material decorated with graphene nanospheres (GNSs). The nanomaterial was drop-coated onto a carbon screen-printed electrode (SPE) to create the GNS-PBA modified electrode (GNS-PBA/SPE). A simple method was used to produce GNS by annealing graphene oxide (GO) solution at high temperature. Field emission scanning electron micrographs confirmed the presence of a spherical shape of GNS with a diameter range of 40-80 nm. A stable and uniform PBA-modified GNS (GNS-PBA) was obtained with a facile ultrasonication step. Thus allowing aminated DNA probes of genetically modified (GM) soybean to be attached to the nanomaterials to form the DNA biosensor. The GNS-PBA/SPE exhibited excellent electrical conductivity via cyclic voltammetry (CV) and differential pulse voltammetry (DPV) tests using potassium ferricyanide (K3[Fe(CN)6]) as the electroactive probe. By employing an anthraquinone monosulfonic acid (AQMS) redox intercalator as the DNA hybridization indicator, the biosensor response was evaluated using the DPV electrochemical method. A good linear relationship between AQMS oxidation peak current and target DNA concentrations from 1.0 × 10-16 to 1.0 × 10-8 M with a limit of detection (LOD) of less than 1.0 × 10-16 M was obtained. Selectivity experiments revealed that the voltammetric GM DNA biosensor could discriminate complementary sequences of GM soybean from non-complementary sequences and hence good recoveries were obtained for real GM soybean sample analysis. The main advantage of using GNS is an improvement of the DNA biosensor analytical performance.
Chili hotness is very much dependent on the concentration of capsaicin present in the chili fruit. A new biosensor based on a horseradish peroxidase enzyme-capsaicin reaction mediated by ferrocene has been successfully developed for the amperometric determination of chili hotness. The amperometric biosensor is fabricated based on a single-step immobilization of both ferrocene and horseradish peroxidase in a photocurable hydrogel membrane, poly(2-hydroxyethyl methacrylate). With mediation by ferrocene, the biosensor could measure capsaicin concentrations at a potential 0.22 V (vs. Ag/AgCl), which prevented potential interference from other electroactive species in the sample. Thus a good selectivity towards capsaicin was demonstrated. The linear response range of the biosensor towards capsaicin was from 2.5-99.0 µM with detection limit of 1.94 µM. A good relative standard deviation (RSD) for reproducibility of 6.4%-9.9% was obtained. The capsaicin biosensor demonstrated long-term stability for up to seven months. The performance of the biosensor has been validated using a standard method for the analysis of capsaicin based on HPLC.
In view of the presence of pathogenic Vibrio cholerae (V. cholerae) bacteria in environmental waters, including drinking water, which may pose a potential health risk to humans, an ultrasensitive electrochemical DNA biosensor for rapid detection of V. cholerae DNA in the environmental sample was developed. Silica nanospheres were functionalized with 3-aminopropyltriethoxysilane (APTS) for effective immobilization of the capture probe, and gold nanoparticles were used for acceleration of electron transfer to the electrode surface. The aminated capture probe was immobilized onto the Si-Au nanocomposite-modified carbon screen printed electrode (Si-Au-SPE) via an imine covalent bond with glutaraldehyde (GA), which served as the bifunctional cross-linking agent. The targeted DNA sequence of V. cholerae was monitored via a sandwich DNA hybridization strategy with a pair of DNA probes, which included the capture probe and reporter probe that flanked the complementary DNA (cDNA), and evaluated by differential pulse voltammetry (DPV) in the presence of an anthraquninone redox label. Under optimum sandwich hybridization conditions, the voltammetric genosensor could detect the targeted V. cholerae gene from 1.0 × 10-17-1.0 × 10-7 M cDNA with a limit of detection (LOD) of 1.25 × 10-18 M (i.e., 1.1513 × 10-13 µg/µL) and long-term stability of the DNA biosensor up to 55 days. The electrochemical DNA biosensor was capable of giving a reproducible DPV signal with a relative standard deviation (RSD) of <5.0% (n = 5). Satisfactory recoveries of V. cholerae cDNA concentration from different bacterial strains, river water, and cabbage samples were obtained between 96.5% and 101.6% with the proposed DNA sandwich biosensing procedure. The V. cholerae DNA concentrations determined by the sandwich-type electrochemical genosensor in the environmental samples were correlated to the number of bacterial colonies obtained from standard microbiological procedures (bacterial colony count reference method).
There is currently no established bacteriological beach quality monitoring (BQM) program in place in Malaysia. To initiate cost-effective, sustainable bacteriological BQM schemes for the ultimate goal of protecting public health, policy decision makers need to be provided robust, indigenous empirical findings that validate appropriate water quality parameters for inclusion in such monitoring programs. This is the first study that assesses the validity of enterococci as an ideal indicator for bacteriological BQM in Malaysia using a multivariate approach. Beach water and sand samples from 7 beach locations were analyzed for a total of twenty-one microbial and non-microbial water quality parameters. A multivariate approach incorporating cluster analyses (CA), principal component analyses (PCA), and factor analysis (FA) was also adopted. Apart from the weak correlations of Staphylococcus aureus with concentrations of Vibro species (r = 0.302, p = 0.037) and total coliforms (r = 0.392, p = 0.006) in seawater, no correlation existed between S. aureus concentration and other parameters. Faecal coliforms failed to correlate with any of the tested parameters. Enterococci also correlated with more quality parameters than faecal coliforms or any other indicator. Multiple linear regressions highlighted a significant, best fit model that could predict enterococci concentrations in relation to other parameters with a maximum predictive success of 69.64%. PCA/FA clearly delineated enterococci and faecal coliforms as parameters that weighed strongly for BQM while Staphylococcus aureus, faecal coliforms and enterococci weighed strongly for beach sand quality monitoring. On the whole, higher correlations of enterococci levels with other parameters than was observed for faecal coliforms suggest that the former be considered a preferred parameter of choice for BQM in Malaysia. Our findings provide meaningful evidence particularly as it relates to the correlation of Enterococci with pathogens and other non-microbial parameters. It also provides empirical data to validate the applicability of the enterococci indicator paradigm for bacteriological beach quality monitoring in Malaysia. The current study thus provides policy decision makers evidenced based approach to parameter streamlining for optimized beach sampling and sustainable bacteriological quality monitoring.
A novel nano-bio composite polypyrrole (PPy)/kappa-carrageenan(KC) was fabricated and characterized for application as a cathode catalyst in a microbial fuel cell (MFC). High resolution SEM and TEM verified the bud-like shape and uniform distribution of the PPy in the KC matrix. X-ray diffraction (XRD) has approved the amorphous structure of the PPy/KC as well. The PPy/KC nano-bio composites were then studied as an electrode material, due to their oxygen reduction reaction (ORR) ability as the cathode catalyst in the MFC and the results were compared with platinum (Pt) as the most common cathode catalyst. The produced power density of the PPy/KC was 72.1 mW/m(2) while it was 46.8 mW/m(2) and 28.8 mW/m(2) for KC and PPy individually. The efficiency of the PPy/KC electrode system is slightly lower than a Pt electrode (79.9 mW/m(2)) but due to the high cost of Pt electrodes, the PPy/KC electrode system has potential to be an alternative electrode system for MFCs.
Novel ionophores comprising various hydroxide and amine structures were immobilized onto poly(vinyl chloride) (PVC) matrices, and these were examined to determine Ti(III) selectivity. To predict the selectivity of Ti(III), a PVC membrane was used to investigate the binding of Ti(III) to c-methylcalix[4]resorcinarene (CMCR). The study showed that the chelating ligand, CMCR, was coordinated selectively to Ti(III) at eight coordination sites involving the oxygen atoms at the interface of the membrane/solution. The membrane was prepared, based on CMCR as an ionophore, sodium tetrakis(4-fluorophenyl) borate (NaTFPB) as a lipophilic ionic additive, and dioctylphthalate (DOP) as a plasticizer. The immobilization of the ionophore and surface characterization studies revealed that the performance of CMCR-immobilized PVC was equivalent to that of mobile ionophores in supported liquid membranes (SLMs). The strengths of the ion-ionophore (CMCR-Ti(OH)(OH(2))(5) (2+)) interactions and the role of ionophores on membranes were studied via UV-Vis, Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and and X-ray diffraction (XRD).
Due to the increasing industrial use of titanium compounds, its determination is the subject of considerable efforts. The ionophore or membrane active recognition is the most important component of any polymeric membrane sensor. The sensor's response depends on the ionophore and bonding between the ionophore and the target ion. Ionophores with molecule-sized dimensions containing cavities or semi-cavities can surround the target ion. The bond between the ionophore and target ion gives different selectivity and sensitivity toward the other ions. Therefore, ionophores with different binding strengths can be used in the sensor.
An optical biosensor based on glutamate dehydrogenase (GLDH) immobilized in a chitosan film for the determination of ammonium in water samples is described. The biosensor film was deposited on a glass slide via a spin-coating method. The ammonium was measured based on beta-nicotinamide adenine dinucleotide (NADH) oxidation in the presence of alpha-ketoglutaric acid at a wavelength of 340 nm. The biosensor showed optimum activity at pH 8. The optimum chitosan concentrations and enzyme loading were found to be at 2% (w/v) and 0.08 mg, respectively. Optimum concentrations of NADH and alpha-ketoglutaric acid both were obtained at 0.15 mM. A linear response of the biosensor was obtained in the ammonium concentration range of 0.005 to 0.5 mM with a detection limit of 0.005 mM. The reproducibility of the biosensor was good, with an observed relative standard deviation of 5.9% (n=8). The biosensor was found to be stable for at least 1 month when stored dry at 4 degrees C.
In the title compound, C13H16N2O2S, the pyrrolidine ring has a twisted conformation on the central -CH2-CH2- bond. Its mean plane is inclined to the 4-meth-oxy-benzoyl ring by 72.79 (15)°. In the crystal, mol-ecules are linked by N-H⋯O and C-H⋯O hydrogen bonds to the same O-atom acceptor, forming chains along [001]. The chains are linked via slipped parallel π-π inter-actions [inter-centroid distance = 3.7578 (13) Å], forming undulating slabs parallel to (100).
In the title compound, C14H18N2O2S, the piperidine ring has a chair conformation. Its mean plane is twisted with respect to the 4-meth-oxy-benzoyl ring, with a dihedral angle of 63.0 (3)°. The central N-C(=S)-N(H)-C(=O) bridge is twisted with an N-C-N-C torsion angle of 74.8 (6)°. In the crystal, mol-ecules are linked by N-H⋯O and C-H⋯O hydrogen bonds, forming chains along the c-axis direction. Adjacent chains are linked by C-H⋯π inter-actions, forming layers parallel to the ac plane. The layers are linked by offset π-π inter-actions [inter-centroid distance = 3.927 (3) Å], forming a supra-molecular three-dimensional structure.
An optical aptasensor-based sensing platform for rapid insulin detection was fabricated. Aminated porous silica microparticles (PSiMPs) were synthesized via a facile mini-emulsion method to provide large surface area for covalent immobilization of insulin-binding DNA aptamer (IGA3) by glutaraldehyde cross-linking protocol. A Nickel-salphen type complex with piperidine side chain [Ni(II)-SP] was synthesized with a simple one-pot reaction, and functionalized as an optical label due to strong π-π interaction between aromatic carbons of G-quadruplex DNA aptamer and planar aromatic groups of Ni(II)-SP to form the immobilized IGA3-Ni(II)-SP complex, i.e. the dye-labeled aptamer, thereby bringing yellow colouration to the immobilized G-quartet plane. Optical characterization of aptasensor towards insulin binding was carried out with a fiber optic reflectance spectrophotometer. The maximum reflectance intensity of the immobilized IGA3-Ni(II)-SP complex at 656 nm decreased upon binding with insulin as aptasensor changed to brownish orange colouration in the background. This allows optical detection of insulin as the colour change of aptasensor is dependent on the insulin concentration. The linear detection range of the aptasensor is obtained from 10 to 50 μIU mL-1 (R2 = 0.9757), which conformed to the normal fasting insulin levels in human with a limit of detection (LOD) at 3.71 μIU mL-1. The aptasensor showed fast response time of 40 min and long shelf life stability of >3 weeks. Insulin detection using healthy human serums with informed consent provided by participants suggests the DNA aptamer biosensor was in good agreement with ELISA standard method using BIOMATIK Human INS (Insulin) ELISA Kit.
An optical genosensor based on Schiff base complex (Zn2+ salphen) DNA label and acrylic microspheres (AMs) as polymer support of the capturing DNA probe (cpDNA) was developed for dengue virus serotype 2 (DEN-2) detection via reflectance spectrophotometric method. The solid-state optical DNA biosensor showed high selectivity and specificity up to one-base mismatch in the target DNA sequence owing to the salphen chemical structure that is rich in localized electrons, and allowed π-π stacking interaction between stacked base pairs of double-stranded DNA (dsDNA). The reflectometric DNA microsensor demonstrated a broad linear detection range towards DEN-2 DNA from 1 × 10-15 M to 1 × 10-3 M with a low limit of detection (LOD) obtained at 1.21 × 10-16 M. The DNA biosensor gave reproducible optical response with a satisfactory relative standard deviation (RSD) at 3.1%, (n = 3), and the reflectance response was stable even after four regeneration cycles of the DNA biosensor. The optical genosensor was proven comparable with standard reverse transcription polymerase chain reaction (RT-PCR) in detecting DEN-2 genome acquired from clinical samples of serum, urine and saliva of dengue virus infected patients under informed consent. The developed reflectometric DNA biosensor is advantageous in offering an early DEN-2 diagnosis, when fever symptom started to manifest in patient.
More than 200 different cultivars of durian exist worldwide but Durio zibethinus or Musang King (MK) is the most premium and prized durian fruit among the recommended varieties. Early identification of this premium variety is critical to protect from non-authentic MK durian cultivars. However, the MK variety's morphological traits are nearly identical to other varieties. Currently, the identification of durian varieties is mostly performed via evaluation of leaf shape, fruit shape, aroma, taste and seed shape and this requires trained personnel for the morphology observation. To enable the rapid identification of the MK variety, PCR amplification of ten durian varieties using six gene candidates from the chloroplast genome was first performed to obtain DNA probes that were specific to the MK durian variety. PCR amplification of ten durian varieties using primers designed confirmed that the nadhA gene sequence showed an obvious difference in the MK variety from other durian varieties. The unique sequence of MK was used as a DNA probe to develop an electrochemical biosensor for the direct identification of the MK durian variety. The electrochemical biosensor was based on the hybridization response of the immobilized DNA probe with the target DNA from the MK variety and was monitored via differential pulse voltammetry technique. Under optimal conditions, the DNA electrochemical biosensor showed a low detection limit at 10% of MK genomic DNA concentration with a wide linear calibration range of 0.05-1.5 µM (R2 = 0.9891) and RSD value of 3.77% (n = 3). The results of the developed DNA biosensor provide high promise for the development of portable sensors employed in the determination of MK variety in the field.
A new silica-gel nanospheres (SiO2NPs) composition was formulated, followed by biochemical surface functionalization to examine its potential in urea biosensor development. The SiO2NPs were basically synthesized based on sol-gel chemistry using a modified Stober method. The SiO2NPs surfaces were modified with amine (-NH2) functional groups for urease immobilization in the presence of glutaric acid (GA) cross-linker. The chromoionophore pH-sensitive dye ETH 5294 was physically adsorbed on the functionalized SiO2NPs as pH transducer. The immobilized urease determined urea concentration reflectometrically based on the colour change of the immobilized chromoionophore as a result of the enzymatic hydrolysis of urea. The pH changes on the biosensor due to the catalytic enzyme reaction of immobilized urease were found to correlate with the urea concentrations over a linear response range of 50-500 mM (R2 = 0.96) with a detection limit of 10 mM urea. The biosensor response time was 9 min with reproducibility of less than 10% relative standard deviation (RSD). This optical urea biosensor did not show interferences by Na+, K+, Mg2+ and NH4+ ions. The biosensor performance has been validated using urine samples in comparison with a non-enzymatic method based on the use of p-dimethylaminobenzaldehyde (DMAB) reagent and demonstrated a good correlation between the two different methods (R2 = 0.996 and regression slope of 1.0307). The SiO2NPs-based reflectometric urea biosensor showed improved dynamic linear response range when compared to other nanoparticle-based optical urea biosensors.
An electrochemical microbiosensor for DNA has been fabricated based on new acrylic microspheres modified with reactive N-acryloxysuccinimide (NAS) functional groups. Hydrophobic poly(n-butylacrylate-N-acryloxysuccinimide) microspheres were synthesized in an emulsion form with a simple one-step photopolymerization technique. Aminated DNA probe was attached to the succinimde functional group of the acrylic microspheres via covalent bonding. The hybridization of the immobilized DNA probe with the complementary DNA was studied by differential pulse voltametry using anthraquninone-2-sulfonic acid monohydrate sodium salt (AQMS) as the electroactive hybridization label. The influences of many factors such as duration of DNA probe immobilization and hybridization, pH, type of ions, buffer concentrations, ionic strength, operational temperature and non-complementary DNA on the biosensor performance were evaluated. Under optimized conditions, the DNA microbiosensor demonstrated a linear response range to target DNA over a wide concentration range of 1.0 × 10(-16) and 1.0 × 10(-8) M with a lower limit of detection (LOD) of 9.46 × 10(-17) M (R(2) = 0.97). This DNA microbiosensor showed good reproducibility with 2.84% RSD (relative standard deviation) (n = 3). Application of the NAS-modified acrylic microspheres in the construction of DNA microbiosensor had improved the overall analytical performance of the resultant DNA microbiosensor when compared with other reported DNA biosensors using other nano-materials for membranes and microspheres as DNA immobilization matrices.
A fluorescence-based fiber optic toxicity biosensor based on genetically modified Escherichia coli (E. coli) with green fluorescent protein (GFP) was developed for the evaluation of the toxicity of several hazardous heavy metal ions. The toxic metals include Cu(II), Cd(II), Pb(II), Zn(II), Cr(VI), Co(II), Ni(II), Ag(I) and Fe(III). The optimum fluorescence excitation and emission wavelengths of the optical biosensor were 400 ± 2 nm and 485 ± 2 nm, respectively. Based on the toxicity observed under optimal conditions, the detection limits of Cu(II), Cd(II), Pb(II), Zn(II), Cr(VI), Co(II), Ni(II), Ag(I) and Fe(III) that can be detected using the toxicity biosensor were at 0.04, 0.32, 0.46, 2.80, 100, 250, 400, 720 and 2600 μg/L, respectively. The repeatability and reproducibility of the proposed biosensor were 3.5%-4.8% RSD (relative standard deviation) and 3.6%-5.1% RSD (n = 8), respectively. The biosensor response was stable for at least five weeks, and demonstrated higher sensitivity towards metal toxicity evaluation when compared to a conventional Microtox assay.
The present research describes a simple method for the identification of the gender of arowana fish (Scleropages formosus). The DNA biosensor was able to detect specific DNA sequence at extremely low level down to atto M regimes. An electrochemical DNA biosensor based on acrylic microsphere-gold nanoparticle (AcMP-AuNP) hybrid composite was fabricated. Hydrophobic poly(n-butylacrylate-N-acryloxysuccinimide) microspheres were synthesised with a facile and well-established one-step photopolymerization procedure and physically adsorbed on the AuNPs at the surface of a carbon screen printed electrode (SPE). The DNA biosensor was constructed simply by grafting an aminated DNA probe on the succinimide functionalised AcMPs via a strong covalent attachment. DNA hybridisation response was determined by differential pulse voltammetry (DPV) technique using anthraquinone monosulphonic acid redox probe as an electroactive oligonucleotide label (Table 1). A low detection limit at 1.0 × 10(-18) M with a wide linear calibration range of 1.0 × 10(-18) to 1.0 × 10(-8) M (R (2) = 0.99) can be achieved by the proposed DNA biosensor under optimal conditions. Electrochemical detection of arowana DNA can be completed within 1 hour. Due to its small size and light weight, the developed DNA biosensor holds high promise for the development of functional kit for fish culture usage.
An ultrasensitive electrochemical biosensor for the determination of pathogenic Vibrio cholerae (V. cholerae) DNA was developed based on polystyrene-co-acrylic acid (PSA) latex nanospheres-gold nanoparticles composite (PSA-AuNPs) DNA carrier matrix. Differential pulse voltammetry (DPV) using an electroactive anthraquninone oligonucleotide label was used for measuring the biosensor response. Loading of gold nanoparticles (AuNPs) on the DNA-latex particle electrode has significantly amplified the faradaic current of DNA hybridisation. Together with the use of a reported probe, the biosensor has demonstrated high sensitivity. The DNA biosensor yielded a reproducible and wide linear response range to target DNA from 1.0 × 10(-21) to 1.0 × 10(-8) M (relative standard deviation, RSD = 4.5%, n = 5) with a limit of detection (LOD) of 1.0 × 10(-21) M (R (2) = 0.99). The biosensor obtained satisfactory recovery values between 91 and 109% (n = 3) for the detection of V. cholerae DNA in spiked samples and could be reused for six consecutive DNA assays with a repeatability RSD value of 5% (n = 5). The electrochemical biosensor response was stable and maintainable at 95% of its original response up to 58 days of storage period.
In this article a luminescence fiber optic biosensor for the microdetection of heavy metal toxicity in waters based on the marine bacterium Aliivibrio fischeri (A. fischeri) encapsulated in alginate microspheres is described. Cu(II), Cd(II), Pb(II), Zn(II), Cr(VI), Co(II), Ni(II), Ag(I) and Fe(II) were selected as sample toxic heavy metal ions for evaluation of the performance of this toxicity microbiosensor. The loss of bioluminescence response from immobilized A. fischeri bacterial cells corresponds to changes in the toxicity levels. The inhibition of the luminescent biosensor response collected at excitation and emission wavelengths of 287 ± 2 nm and 487 ± 2 nm, respectively, was found to be reproducible and repeatable within the relative standard deviation (RSD) range of 2.4-5.7% (n = 8). The toxicity biosensor based on alginate micropsheres exhibited a lower limit of detection (LOD) for Cu(II) (6.40 μg/L), Cd(II) (1.56 μg/L), Pb(II) (47 μg/L), Ag(I) (18 μg/L) than Zn(II) (320 μg/L), Cr(VI) (1,000 μg/L), Co(II) (1700 μg/L), Ni(II) (2800 μg/L), and Fe(III) (3100 μg/L). Such LOD values are lower when compared with other previous reported whole cell toxicity biosensors using agar gel, agarose gel and cellulose membrane biomatrices used for the immobilization of bacterial cells. The A. fischeri bacteria microencapsulated in alginate biopolymer could maintain their metabolic activity for a prolonged period of up to six weeks without any noticeable changes in the bioluminescence response. The bioluminescent biosensor could also be used for the determination of antagonistic toxicity levels for toxicant mixtures. A comparison of the results obtained by atomic absorption spectroscopy (AAS) and using the proposed luminescent A. fischeri-based biosensor suggests that the optical toxicity biosensor can be used for quantitative microdetermination of heavy metal toxicity in environmental water samples.