Balaji Srinivasan

Rapid detection of avian influenza H5N1 virus using impedance measurement of immuno-reaction coupled with RBC amplification.

Avian influenza virus (AIV) subtype H5N1 was first discovered in the 1990 s and since then its emergence has become a likely source of a global pandemic and economic loss. Currently accepted gold standard methods of influenza detection, viral culture and rRT-PCR, are time consuming, expensive and require special training and laboratory facilities. A rapid, sensitive, and specific screening method is needed for in-field or bedside testing of AI virus to effectively implement quarantines and medications. Therefore, the objective of this study was to improve the specificity and sensitivity of an impedance biosensor that has been developed for the screening of AIV H5. Three major components of the developed biosensor are immunomagnetic nanoparticles for the separation of AI virus, a microfluidic chip for sample control and an interdigitated microelectrode for impedance measurement. In this study polyclonal antibody against N1 subtype was immobilized on the surface of the microelectrode to specifically bind AIV H5N1 to generate more specific impedance signal and chicken red blood cells (RBC) were used as biolabels to attach to AIV H5N1 captured on the microelectrode to amplify impedance signal. RBC amplification was shown to increase the impedance signal change by more than 100% compared to the protocol without RBC biolabels, and was necessary for forming a linear calibration curve for the biosensor. The use of a second antibody against N1 offered much greater specificity and reliability than the previous biosensor protocol. The biosensor was able to detect AIV H5N1 at concentrations down to 10(3) EID(50)ml(-1) in less than 2h.

Evaluation study of a portable impedance biosensor for detection of avian influenza virus.

Current methods for detection of avian influenza virus (AIV) based on virus culture and RT-PCR are well established, but they are either time consuming or require specialized laboratory facilities and highly trained technicians. A simple, rapid, robust, and reliable test, suitable for use in the field or at the patients bedside, is urgently needed. In this study, the performance of a newly developed portable impedance biosensor was evaluated by comparison with real-time reverse transcriptase PCR (rRT-PCR) and virus culture for detection of AIV in tracheal and cloacal swab samples collected from experimentally H5N2 AIV infected chickens. The impedance biosensor system was based on a combination of magnetic nanobeads, which were coated with AIV subtype-specific antibody for capture (separation and concentration) of a target virus, and a microfluidic chip with an interdigitated array microelectrode for transfer and detection of target virus, and impedance measurement of the bio-nanobeads and AI virus complexes in a buffer solution. A comparison of results obtained from 59 swab samples using virus culture, impedance biosensor and rRT-PCR methods showed that the impedance biosensor technique was comparable in sensitivity and specificity to rRT-PCR. Detection time for the impedance biosensor is less than 1h.

A Label-free, Microfluidics and Interdigitated Array Microelectrode Based Impedance Biosensor in Combination with Nanoparticles Immunoseparation for Detection of Escherichia coli O157:H7 in Food Samples

A microfluidic flow cell with embedded gold interdigitated array microelectrode (IDAM) was developed and integrated with nanoparticle-antibody conjugates into an impedance biosensor to rapidly detect pathogenic bacteria. The flow cell consisting of a detection microchamber and inlet and outlet microchannels was fabricated by bonding an IDAM chip to a Poly(dimethylsiloxane) (PDMS) microchannel. The detection microchamber with a dimension of 7 x 0.5 x 0.02 mm and a volume of 60 nl was used to collect bacterial cells in the active layer above the electrode. Magnetic nanoparticle antibody conjugates (MNAC) were prepared by conjugating streptavidin-coated magnetic nanoparticles with biotin-labeled polyclonal goat anti-E. coli antibodies and were used in the separation and concentration of target bacteria. The cells of E. coli O157:H7 inoculated in a food sample were first captured by the MNAC, separated and concentrated by applying a magnetic field, washed, and then suspended in mannitol solution and finally injected through the microfluidic flow cell for impedance measurement. The lowest detection limit of this biosensor for detection of E. coli O157:H7 in pure culture and ground beef samples was 8.4 x 105 and 7.9 x 106 cfu ml-1, respectively, and the total detection time from sampling to measurement was 35 min. Equivalent circuit analysis indicated that the bulk medium resistance, double layer capacitance, and dielectric capacitance were responsible for causing the impedance change due to the presence of E. coli O157:H7 cells on the surface of IDAM. Sample pre-enrichment, electrode surface immobilization, and redox probes were not needed in this impedance biosensor.

Insulin detection based on a PDMS microfluidic system

An integrated polydimethysiloxane (PDMS) microfluidic system which is composed of two pneumatic micropumps and one micromixer is developed for high-accuracy detection of insulin. The detection method is based on coupling the highly specific technique of ‘double-antibody sandwich immunoassay’ with the sensitive chemiluminescence of Luminol-Hydrogen Peroxide (H2O2). The immune reactions and other related processes are carried out in the microfluidic system semi automatically. Sample transportation in the microfluidic system is accomplished by two pneumatic PDMS micropumps. Chemiluminescent measurement is conducted in a separate PDMS micromixer using a double-channel syringe pump to inject reagents. Light emitting from this mixer is detected by a highly sensitive photometer when the chemiluminescent regents flow through the mixer chamber. The results indicate that at an actuation pressure of 10psi, a mixer actuation frequency of 5Hz, and an injecting flow rate of 0.5 ml/min, the detection limit of the microfluidic system for insulin is about 10−10 M.

A Microfluidic Filter Chip for Highly Sensitive Chemiluminescence Detection of E. Coli O157:H7

This paper describes a rapid and highly sensitive chemiluminescence detection scheme for E. coli O157:H7 bacteria by using a microfluidic filter chip. The stepped configuration design of the filter chip enables the formation of a monolayer of beads-bacteria-peroxidase labeled antibodies sandwich complex inside the microchamber. The addition of Luminol to the sandwich complex produces a chemiluminescence signal proportional to the concentration of E. coli. The chemiluminescence signal is captured by a fiber optic light guide connected to a luminometer. The monolayer maximizes the optical accessibility of the E. coli cells to the detecting light guide and high chemiluminescence signal strength is achieved. A multi-sampling technique is used to enhance the E. coli capture efficiency by 180%. Due to the combination of the filter chip and the sampling technique, no enrichment scheme is used for detecting extremely low cell concentrations, which is difficult to accomplish with other rapid detection methods.© 2005 ASME

Performance evaluation of a pneumatic-based micromixer for bioconjugation reaction

The bioconjugation efficiency resulting from three PDMS based continuous flow micromixers are experimentally studied at various sample flow rates and pneumatic actuation frequencies. In this study, streptavidin-coupled paramagnetic microbeads and biotin-coupled fluorescent microbeads are separately injected into the micromixers and the resultant binding efficiency is measured by the fluorescence emission of the conjugated beads. The unconjugated beads are removed via magnetic separation. Fluorescence resulting from the conjugation process peaks at 10Hz for each design. Fluorescence resulting from varying the sample flow rate indicates that bioconjugation efficiency peaks at specific flow rates, which vary slightly from design to design. The highest observed efficiency during flow rate tests was observed at 0.8μL/min.

Investigation of a PDMS based micromixer for heterogeneous immunoassays of insulin

An experimental study was carried out to evaluate the performance of a polydimethylsiloxane (PDMS) based micromixer for heterogeneous immunoassays of insulin. The detection method is based on coupling the highly specific double-antibody sandwich immunoassay with the sensitive chemiluminescence of the Luminol-Hydrogen Peroxide reaction. Both the immune and chemiluminescence reactions are conducted in the micromixer, and the resultant light emission is detected by a highly sensitive photometer. The behavior of the luminol based chemiluminescence is parabolic with respect to time, and the maximum light intensity is used here to represent the output of the reaction. The current results indicate that at an actuation pressure of 15psi, an actuation frequency of 30Hz leads to the best reaction result. At these operating conditions, the detection limit of insulin is about 10−14 mol/L while the total reaction time of the insulin immunoassay in the micromixer is less than 5 minutes.

AFM Investigation of Avian Influenza Viruses

The present paper describes a direct label-free diagnostic method that uses atomic force microscopy (AFM) to identify avian influenza virus strains through their electrical properties. In this method, a single virus particle is sandwiched between a rigid, conductive substrate and a conductive AFM tip (radius ∼ 8nm). Electrical characterization is achieved by probing the complex impedance spectrum of the sandwiched virus while mechanical characterization is achieved through nanoindentation. A total of three virus strains (inactivated) with different combinations of glycoprotein subtypes (H2N2, H3N5 and H4N6) were tested. Results from the electrical characterization indicate that the impedance spectra of different virus strains are indeed different. While the average electrical capacitance of a virus particle is about 17pF, the variation from one strain to another can be as high as 70%. A COMSOL Multiphysics™ simulation was carried out to estimate the electrical properties of the glycoproteins on the virus particle by comparing the simulated capacitance to the experimentally obtained values. The result indicates that the electrical conductivity of the glycoproteins is in the range of 9 to 14 mS and the dielectric constant value is around 2. The present results strongly suggest the possibility of using AFM as a diagnostic tool for direct recognition of avian influenza virus strains.Copyright

Microfluidic Filter Chip Based Chemiluminescence Fiber Optic Biosensing Method for the Detection of E. coli O157:H7

A rapid and highly sensitive chemiluminescence biosensor combined with a microfluidic filter chip was investigated and evaluated for the detection of E. coli O157:H7. The microfluidic filter chip consisted of a microchamber and a microchannel glass chips bonded by thermal fusion bonding to form a stepped channel configuration. The filter chip was designed with multiple outlets. The dimension of the microchamber was 1 mm x 1 mm x 11.5 µm. Microchamber was used for the formation of single layer of magnetic beads. The sample containing E. coli O157:H7 was mixed with immunomagnetic microbeads and peroxidase labeled anti-E. coli O157:H7 antibodies to form sandwich complexes. A syringe pump was used to inject the sandwich complexes into the filter chip, and luminol was added to generate a chemiluminescence signal, which was recorded in a real time with a fiber optic light guide connected to a luminometer coupled to data acquisition unit and PC. The result indicated that this filter chip could be used for the detection of as low as 800 CFU/ml of E. coli O157:H7 without enrichment. A multiple enrichment sampling methodology was used for the higher capture of E. coli O157:H7 by the microbeads. We demonstrated that new sampling technique maximized the capture efficiency of magnetic beads and a detection limit of 180 CFU/ml was achieved with an improved sampling technique. The total time of detection was 90 min. Thus, highly sensitive chemiluminescence biosensor combined with the microfluidic filter chip has potential to detect very low cell number of target pathogenic bacteria.