Jianhan Lin

Exploiting Enzyme Catalysis in Ultra-Low Ion Strength Media for Impedance Biosensing of Avian Influenza Virus Using a Bare Interdigitated Electrode

Enzyme catalysis is broadly used in various fields but generally applied in media with high ion strength. Here, we propose the exploitation of enzymatic catalysis in ultra-low ion strength media to induce ion strength increase for developing a novel impedance biosensing method. Avian influenza virus H5N1, a serious worldwide threat to poultry and human health, was adopted as the analyte. Magnetic beads were modified with H5N1-specific aptamer to capture the H5N1 virus. This was followed by binding concanavalin A (ConA), glucose oxidase (GOx), and Au nanoparticles (AuNPs) to create bionanocomposites through a ConA-glycan interaction. The yielded sandwich complex was transferred to a glucose solution to trigger an enzymatic reaction to produce gluconic acid, which ionized to increase the ion strength of the solution, thus decreasing the impedance on a screen-printed interdigitated array electrode. This method took advantages of the high efficiency of enzymatic catalysis and the high susceptibility of electrochemical impedance on the ion strength and endowed the biosensor with high sensitivity and a detection limit of 8 × 10(-4) HAU in 200 μL sample, which was magnitudes lower than that of some analogues based on biosensing methods. Furthermore, the proposed method required only a bare electrode for measurements of ion strength change and had negligible change on the surficial properties of the electrode, though some modification of magnetic beads/Au nanoparticles and the construction of a sandwich complex were still needed. This helped to avoid the drawbacks of commonly used electrode immobilization methods. The merit for this method makes it highly useful and promising for applications. The proposed method may create new possibilities in the broad and well-developed enzymatic catalysis fields and find applications in developing sensitive, rapid, low-cost, and easy-to-operate biosensing and biocatalysis devices.

A sensitive impedance biosensor based on immunomagnetic separation and urease catalysis for rapid detection of Listeria monocytogenes using an immobilization-free interdigitated array microelectrode

In this study, we described a novel impedance biosensor combining immunomagnetic separation with urease catalysis for sensitive detection of foodborne bacteria using Listeria monocytogenes as model and an immobilization-free microelectrode as detector. The monoclonal antibodies (MAbs) were immobilized on the surface of the magnetic nanoparticles (MNPs) with the diameter of 180 nm by biotin-streptavidin system for specifically and efficiently separating Listeria cells from sample background. The polyclonal antibodies (PAbs) and the urease were modified onto the surface of the gold nanoparticles (AuNPs) with the diameter of 20 nm and the modified AuNPs were used to react with Listera to form the MNP-MAb-Listeria-PAb-AuNP-urease sandwich complexes. The urease in the complexes could catalyze the hydrolysis of the urea into ammonium carbonate and this led to an increase in the ionic strength of the media, which could be detected by the microelectrode. The magnetic separation efficiencies for L. monocytogenes at the concentrations ranging from 3.0×10(1) to 3.0×10(4) CFU/mL were over 95% for the pure cultures and over 85% for the spiked lettuce samples. The lower detection limit of this biosensor for L. monocytogenes was found to be 300 CFU/mL in both the pure cultures and the spiked lettuce samples. The microelectrode was demonstrated to be reusable for over 50 times with thorough cleaning by deionized water. This biosensor showed its potential to provide a simple, low-cost and sensitive method for rapid screening of foodborne pathogens and could be extended for detection of other biological or chemical targets.

An impedance immunosensor based on low-cost microelectrodes and specific monoclonal antibodies for rapid detection of avian influenza virus H5N1 in chicken swabs

Early screening of suspected cases is the key to control the spread of avian influenza (AI) H5N1. In our previous studies, an impedance biosensor with an interdigitated array microelectrode based biochip was developed and validated with pure AI H5 virus, but had limitations in cost and reliability of the biochip, specificity of the antibody against Asian in-field H5N1 virus and detection of H5N1 virus in real samples. The purpose of this study is to develop a low-cost impedance immunosensor for rapid detection of Asian in-field AI H5N1 virus in chicken swabs within 1h and validate it with the H5N1 virus. Specific monoclonal antibodies against AI H5N1 virus were developed by fusion of mouse myeloma cells with spleen cells isolated from an H5N1-virus-immunized mouse. Dot-ELISA analysis demonstrated that the developed antibodies had good affinity and specificity with the H5N1 virus. The microelectrodes were redesigned with compact size, fabricated using an improved wet-etching micro-fabrication process with a higher qualified production rate of 70-80%, and modified with the antibodies by the Protein A method. Equivalent circuit analysis indicated that electron transfer resistor was effective with the increase in impedance after capturing of the H5N1 viruses. Linear relationship between impedance change and logarithmic value of H5N1 virus at the concentrations from 2(-1) to 2(4) HAU/50 μl was found and the lower limit of detection was 2(-1) HAU/50 μl. No obvious interferences from non-target viruses such as H6N2, H9N2, Newcastle disease virus, and infectious bronchitis virus were found. Chicken swab tests showed that the impedance immunosensor had a comparable accuracy with real-time RT-PCR compared to viral isolation.

Fast and sensitive detection of foodborne pathogen using electrochemical impedance analysis, urease catalysis and microfluidics.

Early screening of pathogenic bacteria is a key to prevent and control of foodborne diseases. In this study, we developed a fast and sensitive bacteria detection method integrating electrochemical impedance analysis, urease catalysis with microfluidics and using Listeria as model. The Listeria cells, the anti-Listeria monoclonal antibodies modified magnetic nanoparticles (MNPs), and the anti-Listeria polyclonal antibodies and urease modified gold nanoparticles (AuNPs) were incubated in a fluidic separation chip with active mixing to form the MNP-Listeria-AuNP-urease sandwich complexes. The complexes were captured in the separation chip by applying a high gradient magnetic field, and the urea was injected to resuspend the complexes and hydrolyzed under the catalysis of the urease on the complexes into ammonium ions and carbonate ions, which were transported into a microfluidic detection chip with an interdigitated microelectrode for impedance measurement to determine the amount of the Listeria cells. The capture efficiency of the Listeria cells in the separation chip was ∼93% with a shorter time of 30min due to the faster immuno-reaction using the active magnetic mixing. The changes on both impedance magnitude and phase angle were demonstrated to be able to detect the Listeria cells as low as 1.6×10(2)CFU/mL. The detection time was reduced from original ∼2h to current ∼1h. The recoveries of the spiked lettuce samples ranged from 82.1% to 89.6%, indicating the applicability of this proposed biosensor. This microfluidic impedance biosensor has shown the potential for online, automatic and sensitive bacteria separation and detection.

A Label-Free Impedance Immunosensor Using Screen-Printed Interdigitated Electrodes and Magnetic Nanobeads for the Detection of E. coli O157:H7

Escherichia coli O157:H7 is one of the leading bacterial pathogens causing foodborne illness. In this study, an impedance immunosensor based on the use of magnetic nanobeads and screen-printed interdigitated electrodes was developed for the rapid detection of E. coli O157:H7. Magnetic nanobeads coated with anti-E. coli antibody were mixed with an E. coli sample and used to isolate and concentrate the bacterial cells. The sample was suspended in redox probe solution and placed onto a screen-printed interdigitated electrode. A magnetic field was applied to concentrate the cells on the surface of the electrode and the impedance was measured. The impedance immunosensor could detect E. coli O157:H7 at a concentration of 104.45 cfu·mL−1 (~1400 bacterial cells in the applied volume of 25 μL) in less than 1 h without pre-enrichment. A linear relationship between bacteria concentration and impedance value was obtained between 104 cfu·mL−1 and 107 cfu·mL−1. Though impedance measurement was carried out in the presence of a redox probe, analysis of the equivalent circuit model showed that the impedance change was primarily due to two elements: Double layer capacitance and resistance due to electrode surface roughness. The magnetic field and impedance were simulated using COMSOL Multiphysics software.

A microfluidic colorimetric biosensor for rapid detection of Escherichia coli O157:H7 using gold nanoparticle aggregation and smart phone imaging

We intended to develop a novel biosensor using gold nanoparticles (AuNPs) for indicating different concentrations of E. coli O157:H7 and smart phone imaging APP for monitoring color change of the AuNPs. The magnetic nanoparticles (MNPs) modified with the capture antibodies and the polystyrene microspheres (PSs) modified with the detection antibodies and the catalases were simultaneously used to react with the target bacteria in the first mixing channel of the microfluidic chip, and hydrogen peroxide was injected and catalyzed by the catalases on the MNP-bacteria-PS complexes. After the mixture of the AuNPs and the crosslinking agents were injected to react with the catalysate in the second mixing channel and incubated in the detection chamber, the aggregation of the AuNPs was triggered through the crosslinking agents, resulting in the color of the AuNPs changing from blue to red. Finally, the color was measured using the smart phone imaging APP to determine the amount of the bacteria. This biosensor exhibited a good specificity and sensitivity for detection of E. coli O157:H7 in chicken samples with a lower detection limit of 50 CFU/mL.

Tunable, Sheathless Focusing of Diamagnetic Particles in Ferrofluid Microflows with a Single Set of Overhead Permanent Magnets

There has been increasing interest in the use of magnetic fluids to manipulate diamagnetic particles in microfluidic devices. Current methods for diamagnetic-particle focusing in magnetic fluids require either a pair of repulsive magnets or a diamagnetic sheath flow. We demonstrate herein a tunable, sheathless focusing of diamagnetic particles in a microchannel ferrofluid flow with a single set of overhead permanent magnets. Particles are focused into a single stream near the bottom wall of a straight rectangular microchannel, where a magnetic-field minimum is formed as a result of the magnetization of the ferrofluid. This focusing can be readily switched off and on by removing and replacing the permanent magnets. More importantly, the particle-focusing position can be tuned by shifting the magnets with respect to the microchannel. We perform a systematic experimental study of the parametric effects of the fluid-particle-channel system on diamagnetic-particle focusing in terms of a defined particle-focusing effectiveness.