Highly-sensitive organophosphorous pesticide biosensors based on nanostructured films of acetylcholinesterase and CdTe quantum dots

The optical transducer of CdTe semiconductor quantum dots (QDs) has been integrated with acetylcholinesterase enzyme (AChE) by the layer-by-layer (LbL) assembly technique, resulting in a highly sensitive biosensor for detection of organophosphorus pesticides (OPs) in vegetables and fruits based on enzyme inhibition mechanism. The detection limits of the proposed biosensors are as low as 1.05 × 10(-11) M for paraoxon and 4.47 × 10(-12) M for parathion, which are significantly better than those of the conventional GC/MS methods or amperometric biosensors (0.5 nM). These biosensors are used for quick determination of low concentrations of OPs in real vegetable and fruit samples and exhibit satisfactory reproducibility and accuracy. Moreover, the stock stability of the biosensors are very good due to the stabilizing environment for the enzyme in the nanostructures made by LbL technique. Many advantages provided by these biosensors, like fluorescent change recognized by naked eyes and mass production with low cost, will facilitate future development of rapid and high-throughput screening of OPs.     

A screen-printed, amperometric biosensor array incorporated into a novel automated system for the simultaneous determination of organophosphate pesticides

Organophosphate pesticides present serious risks to human and environmental health. A rapid reliable, economical and portable analytical system will be of great benefit in the detection and prevention of contamination. A biosensor array based on six acetylcholinesterase enzymes for use in a novel automated instrument incorporating a neural network program is described. Electrochemical analysis was carried out using chronoamperometry and the measurement was taken 10s after applying a potential of 0 V vs. Ag/AgCl. The total analysis time for the complete assay was less than 6 min. The array was used to produce calibration data with six organophosphate pesticides (OPs) in the concentration range of 10(-5) M to 10(-9) M to train a neural network. The output of the neural network was subsequently evaluated using different sample matrices. There were no detrimental matrix effects observed from water, phosphate buffer, food or vegetable extracts. Furthermore, the sensor system was not detrimentally affected by the contents of water samples taken from each stage of the water treatment process. The biosensor system successfully identified and quantified all samples where an OP was present in water, food and vegetable extracts containing different OPs. There were no false positives or false negatives observed during the evaluation of the analytical system. The biosensor arrays and automated instrument were evaluated in situ in field experiments where the instrument was successfully applied to the analysis of a range of environmental samples. It is envisaged that the analytical system could provide a rapid detection system for the early warning of contamination in water and food.     

Organophosphorus hydrolase multilayer modified microcantilevers for organophosphorus detection

We report a biosensor based on organophosphorus hydrolase (OPH) multilayer modified microcantilever (MCL) for detection of organophosphorus compounds (OPs). The assay is based on substrate-dependent bending of the OPH functionalized MCLs. The cantilever bending amplitude at equilibrium was a function of the concentration of paraoxon with the dynamic range extending from 10(-7) to 10(-3)M. The lower detection limit of approximately 10(-7)M for paraoxon was an order of magnitude better than the OPH-based potentiometric and optical biosensors based on pH modulation. There was a good intra-sensor and an acceptable inter-sensor reproducibility as evidenced by the standard errors of 5% and 15%, respectively. OPs measured using this technique included parathion and diisopropyl fluorophosphate (DFP) in the order of paraoxon>DFP>parathion. The conformational change of the OPH was most likely the main origin of MCL bending.     

Optical detection of choline and acetylcholine based on H₂O₂-sensitive quantum dots

In this paper, we have constructed a simple, rapid and sensitive biosensor for detection of choline and acetylcholine (ACh) based on the hydrogen peroxide (H(2)O(2))-sensitive quantum dots (QDs). The detection limit for choline was 0.1 μM and the linear range was 0.1-0.9 μM and 5-150 μM, respectively. The detection limit for ACh was found to be 10 μM and the linear range was 10-5000 μM. The wide linear ranges were shown to be suitable for routine analyses of choline and ACh. Possible mechanism of the fluorescence of QDs quenched by H(2)O(2) was an electron transfer (ET) process. The experimental conditions of biosensors were optimized, and anti-interference ability was also presented. We also detected the choline in milk samples and the linear range was 5-150 μM. The detection linear range of ACh in serum was 10-140 μM. Most importantly, the recovery of choline in milk and ACh in serum samples were both close to 99%. The excellent performance of this biosensor showed that the method can be used in practice detection of choline and ACh.     

Ambient temperature detection of PCR amplicons with a novel sequence-specific nucleic acid lateral flow biosensor

The spherical porous Pd nanoparticle assemblies (NPAs) have been successfully synthesized by starch-assisted chemical reduction of Pd(II) species at room temperature. Such Pd NPAs are not simply used to enlarge the surface area and to promote the electron transfer. They also catalyze the reduction of H(2)O(2) which are regarded as horseradish peroxidase (HRP) substitutes in electron transfer process. By using them as electrocatalysts, as low as 6.8×10(-7) M H(2)O(2) can be detected with a linear range from 1.0×10(-6) to 8.2×10(-4) M. Moreover, through co-immobilization of such Pd NPAs and glucose oxidase (GOx), a sensitive and selective glucose biosensor is developed. The detection principle lies on measuring the increase of cathodic current by co-reduction of dissolved oxygen and the in situ generated H(2)O(2) during the enzymatic reaction. Under optimal conditions, the detection limit is down to 6.1×10(-6) M with a very wide linear range from 4.0×10(-5) to 2.2×10(-2) M. The proposed biosensor shows a fast response, good stability, high selectivity and reproducibility of serum glucose level. It provides a promising strategy to construct fast, sensitive, stable and anti-interferential amperometric biosensors for early diagnosis and prevention of diabetes.     

Fabrication of a carbon fiber paper as the electrode and its application toward developing a sensitive unmediated amperometric biosensor

Carbon fiber paper (CFP), a material frequently used as the diffusion layer in fuel cells, was found recently to exhibit a potential as an electrode for the development of sensitive, unmediated biosensors. After nitrogen plasma treatment, the CFP exhibited a quasi-reversible behavior to the redox couple (e.g., ferricyanide) with an electron transfer rate constant of 7.2 × 10(-3)cms(-1). This rate constant is approximately double that of a Pt-electrode and is much higher than that of many carbon-based electrodes. The unmediated CFP-based tyrosinase biosensor fabricated for this study exhibited an optimal working potential and operating pH value of -0.2V and 6.5, respectively. Compared to other unmediated tyrosinase biosensors, the CFP-based tyrosinase biosensor offers a high sensitivity for the monitoring of phenolic compounds (17.8, 7.1, 5.2 and 3.7 μA μM(-1)cm(-2) for catechol, phenol, bisphenol and 3-aminophenol, respectively). The lowest detection limit for catechol, phenol, bisphenol and 3-aminophenol was 2, 5, 5 and 12 nM, respectively. Furthermore, this biosensor exhibited a good repeatability, a fast response time (around 10s), and a wide linear dynamic range of detection for phenolic compounds.     

Acetylcholinesterase inhibition-based biosensors for pesticide determination: A review

Pesticides released intentionally into the environment and through various processes contaminate the environment. Although pesticides are associated with many health hazards, there is a lack of monitoring of these contaminants. Traditional chromatographic methods-high-performance liquid chromatography, capillary electrophoresis, and mass spectrometry-are effective for the analysis of pesticides in the environment but have certain limitations such as complexity, time-consuming sample preparation, and the requirement of expensive apparatus and trained persons to operate. Over the past decades, acetylcholinesterase (AChE) inhibition-based biosensors have emerged as simple, rapid, and ultra-sensitive tools for pesticide analysis in environmental monitoring, food safety, and quality control. These biosensors have the potential to complement or replace the classical analytical methods by simplifying or eliminating sample preparation and making field-testing easier and faster with significant decrease in cost per analysis. This article reviews the recent developments in AChE inhibition-based biosensors, which include various immobilization methods, different strategies for biosensor construction, the advantages and roles of various matrices used, analytical performance, and application methods for constructing AChE biosensors. These AChE biosensors exhibited detection limits and linearity in the ranges of 1.0×10(-11) to 42.19 μM (detection limits) and 1.0×10(-11)-1.0×10(-2) to 74.5-9.9×10(3)μM (linearity). These biosensors were stable for a period of 2 to 120days. The future prospects for the development of better AChE biosensing systems are also discussed.     

Gold-coated silica-fiber hybrid materials for application in a novel hydrogen peroxide biosensor

We describe the preparation and characterization of a novel type of core-shell hybrid material for application in a novel hydrogen peroxide biosensor, where the structure consists of a continuous gold shell that encapsulates the silica fiber. The SiO(2)@Au nanofibers had been synthesized by electrospinning silica sol, and then golden seeds were in situ grown on the fiber, lastly the gold-seeded silica fibers were further coated by continuous gold shells. The above nanocomposites had satisfactory chemical stability, excellent biocompatibility and efficient electron transfer property, which may have potential application for the highly sensitive chemical or biological sensors. Cyclic voltammetry (CV) was used to evaluate the electrochemical performance of the SiO(2)@Au nanocomposites at indium tin oxide (ITO). The biosensor showed high sensitivity and fast response upon the addition of H(2)O(2) and the linear range to H(2)O(2) was from 5×10(-6) to 1.0×10(-3)M with a detection limit of 2 μM (S/N=3). The apparent Michaelis-Menten constant of the biosensor was 1.11 mmol L(-1). These results indicated that SiO(2)@Au nanocomposites have potential for constructing of a variety of electrochemical biosensors.     

A novel enzymatic hydrogen peroxide biosensor based on Ag/C nanocables

A novel enzymatic hydrogen peroxide sensor was successfully fabricated based on the nanocomposites containing of Ag/C nanocables and gold nanoparticles (AuNPs). Ag/C nanocables have been synthesized by a hydrothermal method and then AuNPs were assembled on the surface of Ag/C nanocables. The nanocomposites were confirmed by X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM) and energy-dispersive X-ray spectrometry (EDS). The above nanocomposites have satisfactory chemical stability and excellent biocompatibility. Cyclic voltammetry (CV) was used to evaluate the electrochemical performance of the Ag/C/Au nanocomposites at glassy carbon electrode (GCE). The results indicated that the Ag/C/Au nanocomposites exhibited excellent electrocatalytic activity to the reduction of H(2)O(2). It offered a linear range of 6.7×10(-9) to 8.0×10(-6) M, with a detection limit of 2.2×10(-9) M. The apparent Michaelis-Menten constant of the biosensor was 51.7×10(-6) M. These results indicated that Ag/C/Au nanocomposites have potential for constructing of a variety of electrochemical biosensors.     

Gold nanoparticles modified electrode via simple electrografting of in situ generated mercaptophenyl diazonium cations for development of DNA electrochemical biosensor

A novel protocol for development of DNA electrochemical biosensor based on gold nanoparticles (AuNPs) modified glassy carbon electrode (GCE) was proposed, which was carried out by the self-assembly of AuNPs on the mercaptophenyl film (MPF) via simple electrografting of in situ generated mercaptophenyl diazonium cations. The resulting MPF was covalently immobilized on GCE surface via C-C bond with high stability, which was desirable in fabrication of excellent performance biosensors. Probe DNA was self-assembled on AuNPs through the well-known Au-thiol binding. The recognition of fabricated DNA electrochemical biosensor toward complementary single-stranded DNA was determined by differential pulse voltammetry with the use of Co(phen)(3)(3+) as the electrochemical indicator. Taking advantage of amplification effects of AuNPs and stability of MPF, the developed biosensor could detect target DNA with the detection limit of 7.2×10(-11) M, which also exhibits good selectivity, stability and regeneration ability for DNA detection.     

Optical detection of organophosphorus compounds based on Mn-doped ZnSe d-dot enzymatic catalytic sensor

In this paper, we report a sensitive and selective method for detection of organophosphorus compounds (OPs) based on Mn:ZnSe d-dots-enzyme-hydrogen peroxide (H(2)O(2)) fluorescence quenching system. Acetylcholine esterase (AChE) can hydrolyze acetylcholine (ACh) to choline. Subsequently, choline oxidase (ChOx) oxidizes choline to generate H(2)O(2). The enzyme-generated H(2)O(2) can quench the fluorescence of Mn:ZnSe d-dots. When paraoxon are introduced in solution, it can interact with the active centers of AChE and decrease the enzyme activity. This leads to the decrease of the H(2)O(2) production and then the fluorescence quenching rate of Mn:ZnSe d-dots. Experimental results showed that the enzyme inhibition percentage of Mn:ZnSe d-dots-ChOx-AChE-ACh system was proportional to the logarithm of paraoxon in the range 4.84×10(-11) to 4.84×10(-6) mol/L with the detection limit (S/N=3) of 1.31×10(-11) mol/L. The proposed biosensor has been employed for quick determination of paraoxon in tap water and milk samples with satisfactory reproducibility and accuracy. This nano-biosensor was proved to be sensitive, rapid, simple and tolerance of most interfering substances.     

Automated resolution of dichlorvos and methylparaoxon pesticide mixtures employing a Flow Injection system with an inhibition electronic tongue

An amperometric biosensor array has been developed to resolve pesticide mixtures of dichlorvos and methylparaoxon. The biosensor array has been used in a Flow Injection system, in order to operate automatically the inhibition procedure. The sensors used were three screen-printed amperometric biosensors that incorporated three different acetylcholinesterase enzymes: the wild type from Electric eel and two different genetically modified enzymes, B1 and B394 mutants, from Drosophila melanogaster. The inhibition response triplet was modelled using an Artificial Neural Network which was trained with mixture solutions that contain dichlorvos from 10(-4) to 0.1 microM and methylparaoxon from 0.001 to 2.5 microM. This system can be considered an inhibition electronic tongue.     

One-step construction of biosensor based on chitosan-ionic liquid-horseradish peroxidase biocomposite formed by electrodeposition

A simple and controllable electrodeposition approach was established for one-step construction of hydrogen peroxide (H(2)O(2)) biosensors by in situ formation of chitosan-ionic liquid-horseradish peroxidase (CS-IL-HRP) biocomposite film on electrode surface. A highly porous surface with orderly three-dimensional network was revealed by scanning electron microscopy (SEM) investigation. The biocomposite provided improved conductivity and biocompatible microenvironment. The developed biosensor exhibited a fast amperometric response for the determination of H(2)O(2) and 95% of the steady-state current was obtained within 2s. The linear response of the developed biosensor for the determination of H(2)O(2) ranged from 6.0x10(-7) to 1.6x10(-4)M with a detection limit of 1.5x10(-7)M. Performance of the biosensor was evaluated with respect to possible interferences and a good selectivity was revealed. The fabricated biosensor exhibited high reproducibility and long-time storage stability. The ease of the one-step non-manual technique and the promising feature of biocomposite could serve as a versatile platform for the fabrication of electrochemical biosensors.     

An integrated microfluidic system for rapid screening of alpha-fetoprotein-specific aptamers

An electrically neutral cobalt complex, Co(Eim)(4)(NCS)(2) (Eim=1-ethylimidazole, NCS=isothiocyanate) was synthesized and its interaction with double-stranded DNA (dsDNA) was comprehensively studied by electrochemical methods on a glassy carbon electrode (GCE). The experimental results revealed that the cobalt complex could interact with dsDNA via a specific groove-binding mode with an affinity constant of 3.6×10(5)M(-1). The surface-based studies showed that Co(Eim)(4)(NCS)(2) could electrochemically accumulate within the immobilized dsDNA layer rather than single-stranded DNA (ssDNA) layer. Based on this fact, the cobalt complex was utilized as an electrochemical hybridization indicator for the detection of oligonucleotides related to CaMV35S promoter gene. The results showed that the developed biosensor presented very low background interference due to the negligible affinity of the Co(Eim)(4)(NCS)(2) complex with ssDNA. The hybridization specificity experiments further indicated that the biosensor could well discriminate the complementary sequence from the base-mismatched and the non-complementary sequences. The complementary target sequence could be quantified over the range from 5.0×10(-9)M to 2.0×10(-6)M with a detection limit of 2.0×10(-10)M.     

DNA electrochemical biosensor based on thionine-graphene nanocomposite

A novel protocol for development of DNA electrochemical biosensor based on thionine-graphene nanocomposite modified gold electrode was presented. The thionine-graphene nanocomposite layer with highly conductive property was characterized by scanning electron microscopy, transmission electron microscopy, cyclic voltammetry and electrochemical impedance spectroscopy. An amino-substituted oligonucleotide probe was covalently grafted onto the surface of the thionine-graphene nanocomposite by the cross-linker glutaraldehyde. The hybridization reaction on the modified electrode was monitored by differential pulse voltammetry analysis using an electroactive intercalator daunomycin as the indicator. Under optimum conditions, the proposed biosensor exhibited high sensitivity and low detection limit for detecting complementary oligonucleotide. The complementary oligonucleotide could be quantified in a wide range of 1.0 × 10(-12) to 1.0 × 10(-7)M with a good linearity (R(2)=0.9976) and a low detection limit of 1.26 × 10(-13)M (S/N=3). In addition, the biosensor was highly selective to discriminate one-base or two-base mismatched sequences.     

Hydrogen peroxide biosensor based on microperoxidase-11 entrapped in lipid membrane

A highly catalytic activity microperoxidase-11 (MP-11) biosensor for H(2)O(2) was developed to immobilizing the heme peptide in didodecyldimethylammonium bromide (DDAB) lipid membrane. The enzyme electrode thus obtained responded to H(2)O(2) without electron mediator or promoter, at a potential of +0.10 V versus Agmid R:AgCl. A linear calibration curve is obtained over the range from 2.0 x 10(-5) to 2.4 x 10(-3) M. The biosensor responds to hydrogen peroxide in 15 s and has a detection limit of 8 x 10(-7) M (S/N=3) Providing a natural environment with lipid membrane for protein immobilization and maintenance of protein functions is a suitable option for the design of biosensors.     

Electrospun polystyrene-poly(styrene-co-maleic anhydride) nanofiber as a new aptasensor platform

Here we report on a new approach for the electrochemical detection of hydrogen peroxide (H(2)O(2)) based on the co-immobilization of horseradish peroxidase and methylene blue on the functionalized carbon buckypaper supported by a titanium substrate. Cyclic voltammetry was used to study and optimize the performance of the resulting electrochemical biosensor. The proposed biosensor exhibited high analytical performance towards the quantification of H(2)O(2) at the physiological pH 7.4. Under optimized conditions, the biosensor shows a wide linear response range from 0.1 × 10(-6) to 5 × 10(-4)M concentrations of H(2)O(2). The detection limit was determined to be 7.5 × 10(-8)M (based on S/N=3). Reproducibility and stability of the fabricated biosensor were examined with satisfactory results. The biological relevance of the developed electrochemical biosensor has been further studied by the determination of H(2)O(2) in human urine samples of normal volunteers prior to and following the ingestion of coffee. Increased levels of urinary H(2)O(2) concentration suggest that oxidative stress is induced by coffee drinking in humans. There is considerable interest in oxidative stress as relates to human physiology. The sensitive determination of H(2)O(2) in human urine may serve as a valuable biomarker to effectively elucidate specific levels of oxidative stress in vivo.     

Triplex signal amplification for electrochemical DNA biosensing by coupling probe-gold nanoparticles-graphene modified electrode with enzyme functionalized carbon sphere as tracer

An ultrasensitive electrochemical DNA biosensor was constructed by assembling probe labeled gold nanoparticles (ssDNA-AuNP) on electrochemically reduced graphene oxide (ERGO) modified electrode with thiol group tagged (GT) DNA strand (d(GT)(29)SH) and coupling with horseradish peroxidase (HRP) functionalized carbon sphere (CNS) as tracer. The heteronanostructure formed on the biosensor surface appeared relatively good conductor for accelerating the electron transfer, while the HRP tagged CNS provided dual signal amplification for electrochemical biosensing. The triplex signal amplification strategy produced an ultrasensitive electrochemical detection of DNA down to attomolar level (5 aM) with a linear range of 5 orders of magnitude (from 1 × 10(-17)M to 1 × 10(-13)M), and appeared high selectivity to differentiate single-base mismatched and three-base mismatched sequences of DNA. The proposed approach provided a simple and reliable method for DNA detection with high sensitivity and specificity, indicating promising application in bioanalysis and biomedicine.     

Voltammetric biosensors for the determination of formate and glucose-6-phosphate based on the measurement of dehydrogenase-generated NADH and NADPH

This paper describes the development of a modified electrode for the electrocatalytic oxidation of beta-nicotinamide adenine dinucleotide (beta-NADH) and beta-nicotinamide adenine dinucleotide phosphate (beta-NADPH) using electropolymerised 3,4-dihydroxybenzaldehyde (3,4-DHB). Two voltammetric biosensors using enzyme-immobilised membranes were constructed for the determination of formic acid and glucose-6-phosphate (G6P), respectively. The formic acid biosensor based on the combination of formate dehydrogenase (FDH)-modified membrane with 3,4-DHB-coated glassy carbon electrode is one to two orders more sensitive (LOD, 5.0x10(-5) M) than previously reported electrochemical biosensors. Similarly, lower detection limit (4.0x10(-5) M) for the measurement of G6P was achieved using glucose-6-phosphate dehydrogenase (G6PDH) in the presence of beta-NADP(+). The interference of uric acid and ascorbate was minimised by incorporating an additional membrane modified with uricase and ascorbate oxidase, respectively. The biosensing scheme developed in this study can be adopted universally with a number of dehydrogenases for the detection of different substrates.     

A biomimetic olfactory-based biosensor with high efficiency immobilization of molecular detectors

The immobilization efficiency of molecular detectors is of great importance with regard to the performances of biosensors such as the sensitivity, stability, and reproducibility. This paper presents a biomimetic olfactory receptor-based biosensor with better performances by improving the immobilization efficiency of molecular detectors for odorant sensing. A mixed self-assembled monolayers (SAMs) functionalized with specific olfactory receptors (ODR-10) was constructed on the sensitive area of surface acoustic wave (SAW) chip. The immobilization of ODR-10 was characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The responses of this biosensor to various odorants were recorded by monitoring the resonance frequency shifts of SAW, which is correlated to the mass loading on its sensitive area. All the results demonstrate this biosensor can specifically respond to the natural ligand of ODR-10, diacetyl, with high sensitivity and stability. The sensitivity is 4 kHz/ng, which is 2× higher than that of previous work. The detection limit is 1.2×10(-11) mM. The major advances on immobilization efficiency of molecular detectors presented in this work could substantially promote and accelerate the researches and applications of olfactory receptor-based biosensors with different transducers, such as quartz crystal microbalance (QCM), surface plasma resonance (SPR), and field effect transistors (FET).