Label-free reflectometric interference microchip biosensor based on nanoporous alumina for detection of circulating tumour cells

In this report, a label-free reflectometric interference spectroscopy (RIfS) based microchip biosensor for the detection of circulating tumour cells (CTCs) is demonstrated. Highly ordered nanoporous anodic aluminium oxide (AAO) fabricated by electrochemical anodization of aluminium foil was used as the RIfS sensing platform. Biotinylated anti-EpCAM antibody that specifically binds to human cancer cells of epithelial origin such as pancreatic cancer cells (PANC-1) was covalently attached to the AAO surface through multiple surface functionalization steps. Whole blood or phosphate buffer saline spiked with low numbers of pancreatic cancer cells were successfully detected by specially designed microfluidic device incorporating an AAO RIfS sensor, without labour intensive fluorescence labelling and/or pre-enhancement process. Our results show that the developed device is capable of selectively detecting of cancer cells, within a concentrations range of 1000-100,000 cells/mL, with a detection limit of <1000 cells/mL, a response time of <5 min and sample volume of 50 μL of. The presented RIfS method shows considerable promise for translation to a rapid and cost-effective point-of-care diagnostic device for the detection of CTCs in patients with metastatic cancer.     

Development of a quantum dot molecularly imprinted polymer sensor for fluorescence detection of atrazine

Atrazine is a common agricultural pesticide which has been reported to occur widely in surface drinking water, making it an environmental pollutant of concern. In the quest for developing sensitive detection methods for pesticides, the use of quantum dots (QDs) as sensitive fluorescence probes has gained momentum in recent years. QDs have attractive and unique optical properties whilst coupling of QDs to molecularly imprinted polymers (MIPs) has been shown to offer excellent selectivity. Thus, the development of QD@MIPs based fluorescence sensors could provide an alternative for monitoring herbicides like atrazine in water. In this work, highly fluorescent CdSeTe/ZnS QDs were fabricated using the conventional organometallic synthesis approach and were then encapsulated with MIPs. The CdSeTe/ZnS@MIP sensor was characterized and applied for selective detection of atrazine. The sensor showed a fast response time (5 min) upon interaction with atrazine and the fluorescence intensity was linearly quenched within the 2–20 mol L^?1 atrazine range. The detection limit of 0.80 × 10^?7 mol L^?1 is comparable to reported environmental levels. Lastly, the sensor was applied in real water samples and showed satisfactory recoveries (92–118%) in spiked samples, hence it is a promising candidate for use in water monitoring.     

Highly stable electrochemical immunosensor for carcinoembryonic antigen

The long-term stability of sensing interfaces is an important issue in biosensor fabrication. A novel stable gold nanoparticle (AuNP)-modified glassy carbon (GC) electrode interface (GC-Ph-AuNP)-based biosensor for detecting carcinoembryonic antigen (CEA) was developed. GC electrodes were modified with 1,4-phenylenediamine to form a stable layer, and then AuNPs were bound onto the GC electrodes through CAu bonds. Anti-CEA was directly adsorbed on AuNPs fixed on the GC electrode. The linear range of the immunosensor was from 10 fg to 100 ng mL(-1) with a detection limit of 3 fg mL(-1) (S/N=3). The current of the immunosensor was increased by 4% after one month. The GC-Ph-AuNP immunosensor showed high sensitivity, a wide linear range, low detection limit, and good selectivity and stability. The immobilization method of the immunosensor could be widely applied to construct other immunosensors.     

Multi-analyte assay for triazines using cross-reactive antibodies and neural networks

A biosensor system based on total internal reflectance fluorescence (TIRF) was used to discriminate a mixture of the triazines atrazine and simazine. Only cross-reactive antibodies were available for these two analytes. The biosensor is fully automated and can be regenerated allowing several hundreds of measurements without any user input. Even a remote control for online monitoring in the field is possible. The multivariate calibration of the sensor signal was performed using artificial neural networks, as the relationship between the sensor signals and the concentration of the analytes is highly non-linear. For the development of a multi-analyte immunoassay consisting of two polyclonal antibodies with cross-reactivity to atrazine and simazine and different derivatives immobilised on the transducer surface, the binding characteristics between these substances like binding capacity and cross-reactivity were characterised. The examination of three different measurement procedures showed that a two-step measurement using only one antibody per step allows a quantification of both analytes in a mixture with limits of detection of 0.2 microg/l for atrazine and 0.3 microg/l for simazine. The biosensor is suitable for online monitoring in the field and remote control is possible.     

Silicon photonic crystal nanocavity-coupled waveguides for error-corrected optical biosensing

A photonic crystal (PhC) waveguide based optical biosensor capable of label-free and error-corrected sensing was investigated in this study. The detection principle of the biosensor involved shifts in the resonant mode wavelength of nanocavities coupled to the silicon PhC waveguide due to changes in ambient refractive index. The optical characteristics of the nanocavity structure were predicted by FDTD theoretical methods. The device was fabricated using standard nanolithography and reactive-ion-etching techniques. Experimental results showed that the structure had a refractive index sensitivity of 10(-2) RIU. The biosensing capability of the nanocavity sensor was tested by detecting human IgG molecules. The device sensitivity was found to be 2.3±0.24×10(5) nm/M with an achievable lowest detection limit of 1.5 fg for human IgG molecules. Additionally, experimental results demonstrated that the PhC devices were specific in IgG detection and provided concentration-dependent responses consistent with Langmuir behavior. The PhC devices manifest outstanding potential as microscale label-free error-correcting sensors, and may have future utility as ultrasensitive multiplex devices.     

Disposable TSM-biosensor based on viscosity changes of the contacting medium

The thickness shear mode (TSM)-sensor responds to changes of mechanical properties of the material contacting the surface of the sensor. One of the material properties is the viscosity of a liquid. Abiosensor based on the TSM-resonator for the detection of endotoxin has been developed. It exploits the viscosity–density change during the reaction of endotoxin with limulus amebocyte lysate (LAL). The effect of surface properties of the sensor has been investigated to achieve better output signals. It is shown that the sensor requires a hydrophilic surface to get a better coupling between the sensor and the LAL–endotoxin solution. The TSM biosensor is able to detect an endotoxin concentration as low as 100 fg/ml by using only 50-μl standard LAL solution. The disadvantages of reusable sensors, such as the contamination from previous measurement of endotoxin and the cost of the regeneration or reclining processes of the sensor, have been eliminated by using a cost effective disposable TSM-sensor.     

Comparison of colorimetric and chemiluminescent enzyme‐linked immunosorbent assay for the detection of endosulfan in food samples

Pesticides have become part of food protection since their inception. Endosulfan, an organochlorine insecticide, has been used against insect pests such as whiteflies, aphids, red spiders and mites. Methods of immunochemical assays have been devised for the determination and analysis of pesticides and commonly used for the analysis of contaminants in food, water, soil and body fluids. Chicken IgY antibodies raised against endosulfan haptens were used for the detection of endosulfan. We have compared colorimetric (CO) and chemiluminescence (CL) enzyme‐linked immunosorbent assay (ELISA) techniques for the detection of endosulfan isomers in a food matrix. CL ELISA assay was found to be more sensitive than CO assay. The mean recovery was 81.2–95.6% for α‐ and β‐endosulfan‐spiked food samples with 2.8–4.6% relative standard deviation. The detection of the endosulfan isomers was linear in the range 100 µg/mL–5 fg/mL, with a limit of detection at 100 µg/mL and 5 fg/mL for the CL ELISA method and 100 µg/mL and 1 ng/mL for the CO ELISA method respectively. These methods can be used for the rapid and reliable detection of organochlorine pesticide endosulfan. Copyright © 2015 John Wiley & Sons, Ltd.     

Rapid detection of atrazine and its metabolite in raw urine by extractive electrospray ionization mass spectrometry

Herbicides such as atrazine are widely used in the biosphere. Urine analysis is usually performed to evaluate the toxicological effects associated with atrazine exposure. A simple procedure based on the extractive electrospray ionization mass spectrometry (EESI-MS) method was established to detect atrazine and its metabolites in undiluted raw urine without sample pretreatment. A 4.3 × 10⁻¹⁴ g atrazine in spiked raw urine was detected and identified by EESI/MS/MS/MS. The detection limit was found to be 0.4 fg for atrazine (m/z 174) and 0.2 fg for 2-chloro-4, 6-diamino-S-triazine (DACT) (m/z 129) (S/N = 3) in EESI/MS/MS. A linear dynamic range of 4–5 orders of magnitude (r = 0.996) was determined for both atrazine and DACT. A single sample analysis was completed using tandem EESI-MS/MS within 1 min, providing a practical convenient method for rapid analysis of trace amounts of targeted metabolites present in complex matrices. Thus, tandem EESI-MS is potentially useful for previously discovered biomarker detection in multiple applications such as clinical diagnosis, drug discovery and forensic science.     

High-performance electrochemical biosensor for the detection of total cholesterol

We report on a highly sensitive electrochemical biosensor for the determination of total cholesterol. The novel biosensor was fabricated by co-immobilizing three enzymes, cholesterol oxidase (ChO(x)), cholesterol esterase (ChE) and horseradish peroxidase (HRP), on nanoporous gold networks directly grown on a titanium substrate (Ti/NPAu/ChO(x)-HRP-ChE). The morphology and composition of the fabricated nanoporous gold were characterized by scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS) and X-ray diffraction spectroscopy (XRD). The electrochemical behaviour of the Ti/NPAu/ChO(x)-HRP-ChE biosensor was studied using cyclic voltammetry (CV), showing that the developed biosensor possessed high selectivity and high sensitivity (29.33 μA mM?1 cm?2). The apparent Michaelis-Menten constant, K(M)(app) of this biosensor was very low (0.64 mM), originating from the effective immobilization process and the nanoporous structure of the substrate. The biosensor exhibited a wide linear range up to 300 mg dL?1 in a physiological condition (pH 7.4), which makes it very promising for the clinical determination of cholesterol. The fabricated biosensor was further tested using real food samples margarine, butter and fish oil, showing that the biosensor has the potential to be used as a facile cholesterol detection tool in food and supplement quality control.     

Ultrasensitive cDNA detection of dengue virus RNA using electrochemical nanoporous membrane-based biosensor

A nanoporous alumina membrane-based ultrasensitive DNA biosensor is constructed using 5'-aminated DNA probes immobilized onto the alumina channel walls. Alumina nanoporous membrane-like structure is carved over platinum wire electrode of 76 μm diameter dimension by electrochemical anodization. The hybridization of complementary target DNA with probe DNA molecules attached inside the pores influences the pore size and ionic conductivity. The biosensor demonstrates linear range over 6 order of magnitude with ultrasensitive detection limit of 9.55×10(-12) M for the quantification of ss-31 mer DNA sequence. Its applicability is challenged against real time cDNA PCR sample of dengue virus serotype1 derived from asymmetric PCR. Excellent specificity down to one nucleotide mismatch in target DNA sample of DENV3 is also demonstrated.     

Piezoelectric urea biosensor based on immobilization of urease onto nanoporous alumina membranes

The urease was immobilized onto nanoporous alumina membranes prepared by the two-step anodization method, and a novel piezoelectric urea sensing system with separated porous alumina/urease electrode has been developed through measuring the conductivity change of immobilized urease/urea reaction. The process of urease immobilization was optimized and the performance of the developed urea biosensor was evaluated. The obtained urea biosensor presented high-selectivity monitoring of urea, better reproducibility (S.D. = 0.02, n = 6), shorter response time (30 s), wider linear range (0.5 μM to 3 mM), lower detection limit (0.2 μM) and good long-term storage stability (with about 76% of the enzymatic activity retained after 30 days). The clinical analysis of the urea biosensor confirmed the feasibility of urea detection in urine samples.     

Controlled immobilization of acetylcholinesterase on improved hydrophobic gold nanoparticle/Prussian blue modified surface for ultra-trace organophosphate pesticide detection

An ultrasensitive amperometric acetylcholinesterase (AChE) biosensor was fabricated by controlled immobilization of AChE on gold nanoparticles/poly(dimethyldiallylammonium chloride) protected Prussian blue (Au-PDDA-PB) nanocomposite modified electrode surface for the detection of organophorous pesticide. The Au-PDDA-PB membrane served as an excellent matrix for the immobilization of enzyme, which not only enhanced electron transfer but also possessed a relatively large surface area. In addition, the surface hydrophilicity of the Au-PDDA-PB nanocomposite was finely controlled in the static water contact angle range of 25.6-78.1° by adjusting the ratio of gold nanoparticles to PDDA-PB. On an optimized hydrophobic surface, the AChE adopts an orientation with both good activity and stability, which has been proven by electrochemical methods. Benefit from the advantages of the Au-PDDA-PB nanocomposite and the good activity and stability of AChE, the biosensor shows significantly improved sensitivity to monocrotophos, a typical highly toxic organophorous pesticide, with wide linear range (1.0-1000 pg/mL and 1.0-10 ng/mL) and an ultra-low detection limit of 0.8 pg/mL. The biosensor exhibits accuracy, good reproducibility and stability. This strategy may therefore provide useful information for the controlled immobilization of protein and the design of highly sensitive biosensors.     

Direct triazine herbicide detection using a self-assembled photosynthetic reaction center from purple bacterium

In this study, a direct detection system for triazine derivative herbicides was developed using the photosynthetic reaction center (RC) from the purple bacterium,Rhodobacter sphaeroides, and surface plasmon resonance (SPR) apparatus. The histidine-tagged RCs were immobilized on an SPR gold chip using nickel-nitrilotriacetic acid groups as a binder for one of the triazine herbicide, atrazine. The SPR responses were proportional to the sample concentrations of atrazine in the range 0.1–1 μg/mL. The sensitivity of the direct detection of atrazine using the RC-assembled sensor chip was higher than that using the antibody-immobilized chip. The other types of herbicides, DCMU or MCPP, were not detected with such high sensitivity. The results indicated the high binding selectivity of the RC complex.     

Optical microsensors for pesticides identification based on porous silicon technology

A simple and low cost optical sensor, based on porous silicon nanotechnology, has been used to detect and quantify the presence of atrazine pesticide in water and humic acid solutions. In both cases, a well defined optical signal variation can be registered, even at low concentration as 1 ppm. The phenomenon can be ascribed to the capillary infiltration of liquid into the pores, which changes the average refractive index of the structure. Due to the resonant cavity enhanced operation of the proposed sensors, very low detection limits can be reached.     

Removal of prioritary pesticides contamining r'mel ground water by using organic waste residues

This study evaluated pesticide contamination of R'mel ground water located in northwest Morocco. The study area is densely populated and thriving, with intensive agriculture. Various techniques, including stir bar sorptive extraction (SBSE) and gas chromatography with mass spectroscopy detection (GC-MS), were used for the quantitative determination of 13 pesticides including alachlor, aldrin, atrazine, chlorpyrifos, chlorfenvinphos, dieldrin, alpha-endosulfan, endrin, hexachlorobenzene, beta-HCH, gamma-HCH (lindane), simazine and trifluralin. The survey results showed that contamination by pesticide residues is widespread in the area. With the exception of atrazine, the average concentrations were all below the regulatory limits established by the European Union. The potential of ten natural organic substances to eliminate pesticides included in the European Water Framework Directive was evaluated. The absorbents with the highest removal efficiency were date and olives stones and, to a Lesser degree, Raphanus raphanistrum and Cistus ladaniferus. The adsorption tests gave very satisfying results and pointed to the possible application of these supports as ecoLogical remediation techniques to prevent pesticide pollution of aquatic ecosystems.     

Label-free and high-sensitive detection of Salmonella using a surface plasmon resonance DNA-based biosensor

A method based on surface plasmon resonance (SPR) DNA biosensor has been developed for label-free and high-sensitive detection of Salmonella. A biotinylated single-stranded oligonucleotide probe was designed to target a specific sequence in the invA gene of Salmonella and then immobilized onto a streptavidin coated dextran sensor surface. The invA gene was isolated from bacterial cultures and amplified using a modified semi-nested asymmetric polymerase chain reaction (PCR) technique. In order to investigate the hybridization detection, experiments with different concentration of synthetic target DNA sequences have been performed. The calibration curve of synthetic target DNA had good linearity from 5 nM to 1000 nM with a detection limit of 0.5 nM. The proposed method was applied successfully to the detection of single-stranded invA amplicons from three serovars of Salmonella, i.e., Typhimurium, Enterica and Derby, and the responses to PCR products were related to different S. typhimurium concentrations in the range from 10(2) to 10(10) CFU mL(-1). While with this system to detect E. coli and S. aureus, no significant signal was observed, demonstrating good selectivity of the method. In addition, the hybridization can be completed within 15 min, and the excellent sensor surface regeneration allows at least 300 assay cycles without obvious loss of performance.     

A sensitive and regenerable biosensor for organophosphate pesticide based on self‐assembled multilayer film with CdTe as fluorescence probe

A fluorescence biosensor for organophosphorus pesticides was developed. A pH indicator, CdTe quantum dots, were used as an optical transducer of the inhibition of enzyme by analyte. Through the intervening agency of chitosan, the recognition elements (acetylcholinesterase and CdTe) were immobilized onto the surface of quartz by electrostatic attraction to form a self‐assembled multilayer film. In the absence of pesticide, acetylcholine was biocatalytically hydrolysed to yield acetic acid and choline. The released acid resulted in pH decrease, which was sensed by the immobilized pH indicator (CdTe). In the presence of pesticide, the action of acetylcholine was reduced; the fluorescence intensity of the film changed and was related to the concentration of pesticide. This multilayer film could be used as the biosensor for monocrotophos, with a detection limit of 3.20 × 10^?8 mol/L; the sensitivity was 100 times higher than that of CdTe in aqueous solution. The sensor was easily regenerated, and had good stability and selectivity for organophosphorus pesticides. Copyright © 2011 John Wiley & Sons, Ltd.     

RAPID DETECTION OF SALMONELLA TYPHIMURIUM IN FOOD SAMPLES USING A BIENZYME ELECTROCHEMICAL BIOSENSOR WITH FLOW INJECTION

A bienzyme (tyrosinase and horseradish peroxidase) electrochemical biosensor was developed for detection of Salmonella typhimurium, and evaluated for application in a flow injection system coupled with immunomagnetic separation for food samples. Parameters for immunomagnetic separation, enzymatic reaction, flow injection and electrochemical detection were determined using pure culture samples. The selectivity was tested in the presence of Listeria monocytogenes, Campylobacter jejuni and E. coli 0157:H7. The results showed a linear relationship for logarithmic values between peak current ratio and the cell number of S. typhimurium in the range of 10³ 10⁵ cfu/mL, with R²= 0.99. The detection limit of this method was 1.09 × 10³ cfu/mL for S. typhimurium and the detection time was 2.5 h. Samples of chicken carcass wash water and ground beef were used to evaluate the biosensor. The results demonstrated that this biosensor has a potential for rapid detection of different pathogens in various food samples.     

A potentiometric sensor designed on the basis of urease immobilized in polyelectrolyte microcapsules

A potentiometric biosensor has been designed on the basis of glass pH-electrode with a sensing device of the microcellular polyelectrolytic coating containing urease. The polymeric walls of the coating are readily permeable for low-molecular weight compounds, including urea, but are impermeable for macromolecules. The main characteristics of the biosensor in various experimental solutions containing urea, low-molecular-weight salt, and buffer have been obtained. The sensor has been shown to be stable for at least three weeks. The standard curves of the sensor are linear in the range of urea concentrations from 0.2 to 20 mM.     

Multiplexed detection of cardiac biomarkers in serum with nanowire arrays using readout ASIC

Early detection of cardiac biomarkers for diagnosis of heart attack is the key to saving lives. Conventional method of detection like the enzyme-linked immunosorbent assay (ELISA) is time consuming and low in sensitivity. Here, we present a label-free detection system consisting of an array of silicon nanowire sensors and an interface readout application specific integrated circuit (ASIC). This system provides a rapid solution that is highly sensitive and is able to perform direct simultaneous-multiplexed detection of cardiac biomarkers in serum. Nanowire sensor arrays were demonstrated to have the required selectivity and sensitivity to perform multiplexed detection of 100 fg/ml troponin T, creatine kinase MM, and creatine kinase MB in serum. A good correlation between measurements from a probe station and the readout ASIC was obtained. Our detection system is expected to address the existing limitations in cardiac health management that are currently imposed by the conventional testing platform, and opens up possibilities in the development of a miniaturized device for point-of-care diagnostic applications.