DNA-Mediated Charge Transport Devices for Protein Detection

Detection of biologically relevant targets, including small molecules, proteins, DNA, and RNA, is vital for fundamental research as well as clinical diagnostics. Sensors with biological elements provide a natural foundation for such devices because of the inherent recognition capabilities of biomolecules. Electrochemical DNA platforms are simple, sensitive, and do not require complex target labeling or expensive instrumentation. Sensitivity and specificity are added to DNA electrochemical platforms when the physical properties of DNA are harnessed. The inherent structure of DNA, with its stacked core of aromatic bases, enables DNA to act as a wire via DNA-mediated charge transport (DNA CT). DNA CT is not only robust over long molecular distances of at least 34 nm, but is also especially sensitive to anything that perturbs proper base stacking, including DNA mismatches, lesions, or DNA-binding proteins that distort the π-stack. Electrochemical sensors based on DNA CT have previously been used for single-nucleotide polymorphism detection, hybridization assays, and DNA-binding protein detection. Here, improvements to (i) the structure of DNA monolayers and (ii) the signal amplification with DNA CT platforms for improved sensitivity and detection are described.

First, improvements to the control over DNA monolayer formation are reported through the incorporation of copper-free click chemistry into DNA monolayer assembly. As opposed to conventional film formation involving the self-assembly of thiolated DNA, copper-free click chemistry enables DNA to be tethered to a pre-formed mixed alkylthiol monolayer. The total amount of DNA in the final film is directly related to the amount of azide in the underlying alkylthiol monolayer. DNA monolayers formed with this technique are significantly more homogeneous and lower density, with a larger amount of individual helices exposed to the analyte solution. With these improved monolayers, significantly more sensitive detection of the transcription factor TATA binding protein (TBP) is achieved.

Using low-density DNA monolayers, two-electrode DNA arrays were designed and fabricated to enable the placement of multiple DNA sequences onto a single underlying electrode. To pattern DNA onto the primary electrode surface of these arrays, a copper precatalyst for click chemistry was electrochemically activated at the secondary electrode. The location of the secondary electrode relative to the primary electrode enabled the patterning of up to four sequences of DNA onto a single electrode surface. As opposed to conventional electrochemical readout from the primary, DNA-modified electrode, a secondary microelectrode, coupled with electrocatalytic signal amplification, enables more sensitive detection with spatial resolution on the DNA array electrode surface. Using this two-electrode platform, arrays have been formed that facilitate differentiation between well-matched and mismatched sequences, detection of transcription factors, and sequence-selective DNA hybridization, all with the incorporation of internal controls.

For effective clinical detection, the two working electrode platform was multiplexed to contain two complementary arrays, each with fifteen electrodes. This platform, coupled with low density DNA monolayers and electrocatalysis with readout from a secondary electrode, enabled even more sensitive detection from especially small volumes (4 μL per well). This multiplexed platform has enabled the simultaneous detection of two transcription factors, TBP and CopG, with surface dissociation constants comparable to their solution dissociation constants.

With the sensitivity and selectivity obtained from the multiplexed, two working electrode array, an electrochemical signal-on assay for activity of the human methyltransferase DNMT1 was incorporated. DNMT1 is the most abundant human methyltransferase, and its aberrant methylation has been linked to the development of cancer. However, current methods to monitor methyltransferase activity are either ineffective with crude samples or are impractical to develop for clinical applications due to a reliance on radioactivity. Electrochemical detection of methyltransferase activity, in contrast, circumvents these issues. The signal-on detection assay translates methylation events into electrochemical signals via a methylation-specific restriction enzyme. Using the two working electrode platform combined with this assay, DNMT1 activity from tumor and healthy adjacent tissue lysate were evaluated. Our electrochemical measurements revealed significant differences in methyltransferase activity between tumor tissue and healthy adjacent tissue.

As differential activity was observed between colorectal tumor tissue and healthy adjacent tissue, ten tumor sets were subsequently analyzed for DNMT1 activity both electrochemically and by tritium incorporation. These results were compared to expression levels of DNMT1, measured by qPCR, and total DNMT1 protein content, measured by Western blot. The only trend detected was that hyperactivity was observed in the tumor samples as compared to the healthy adjacent tissue when measured electrochemically. These advances in DNA CT-based platforms have propelled this class of sensors from the purely academic realm into the realm of clinically relevant detection.

Interfacial immobilization of monoclonal antibody and detection of human prostate-specific antigen.

Antibody orientation and its antigen binding efficiency at interface are of particular interest in many immunoassays and biosensor applications. In this paper, spectroscopic ellipsometry (SE), neutron reflection (NR), and dual polarization interferometry (DPI) have been used to investigate interfacial assembly of the antibody [mouse monoclonal anti-human prostate-specific antigen (anti-hPSA)] at the silicon oxide/water interface and subsequent antigen binding. It was found that the mass density of antibody adsorbed at the interface increased with solution concentration and adsorption time while the antigen binding efficiency showed a steady decline with increasing antibody amount at the interface over the concentration range studied. The amount of antigen bound to the interfacial immobilized antibody reached a maximum when the surface-adsorbed amount of antibody was around 1.5 mg/m(2). This phenomenon is well interpreted by the interfacial structural packing or crowding. NR revealed that the Y-shaped antibody laid flat on the interface at low surface mass density with a thickness around 40 Å, equivalent to the short axial length of the antibody molecule. The loose packing of the antibody within this range resulted in better antigen binding efficiency, while the subsequent increase of surface-adsorbed amount led to the crowding or overlapping of antibody fragments, hence reducing the antigen binding due to the steric hindrance. In situ studies of antigen binding by both NR and DPI demonstrated that the antigen inserted into the antibody layer rather than forming an additional layer on the top. Stability assaying revealed that the antibody immobilized at the silica surface remained stable and active over the monitoring period of 4 months. These results are useful in forming a general understanding of antibody interfacial behavior and particularly relevant to the control of their activity and stability in biosensor development.

KNOCK DETECTION BY AUDIBLE, ACCELEROMETER, PRESSURE AND IONIC CURRENT METHODS.

This paper describes the ways which are used at the present time by the Ford Motor Company for the detection of knock. A description of the available methods is given, and their relative effectiveness is assessed by analysing data taken simultaneously by all of these methods. It is shown that no one technique has an overwhelming advantage, but that each technique has its individual merits and demerits.

Fibre optic distributed sensor system for condition monitoring of synthetic ropes

Reliability of the measuring devices is very important problem. Optical fibre sensors are very efficient. The use of optical fibre sensors for monitoring the physical and chemical parameters has been expanding over resent years. These sensors are applied for monitoring the structural integrity of long, parallel lay synthetic ropes. Such ropes are corrosion free, however, their operational lifetime under cyclic load is not well understood and premature failure can occur due to slippage and breakage of yarns within the rope. The monitoring system has been proposed which is based on acoustic detection of yarn breakage. Monitoring the strain and temperature is performed using the array of fibre gratings distributed along the rope length.

Towards real-time profiling of sprints using wearable pressure sensors

On-body sensor systems for sport are challenging since the sensors must be lightweight and small to avoid discomfort, and yet robust and highly accurate to withstand and capture the fast movements associated with sport. In this work, we detail our experience of building such an on-body system for track athletes. The paper describes the design, implementation and deployment of an on-body sensor system for sprint training sessions. We autonomously profile sprints to derive quantitative metrics to improve training sessions. Inexpensive Force Sensitive Resistors (FSRs) are used to capture foot events that are subsequently analysed and presented back to the coach. We show how to identify periods of sprinting from the FSR data and how to compute metrics such as ground contact time. We evaluate our system using force plates and show that millisecond-level accuracy is achievable when estimating contact times. © 2012 Elsevier B.V. All rights reserved.

Graphene SOI CMOS sensors for detection of PPB levels of NO2 in air

There is considerable demand for sensors that are capable of detecting ultra-low concentrations (sub-PPM) of toxic gases in air. Of particular interest are NO2 and CO that are exhaust products of internal combustion engines. Electrochemical (EC) sensors are widely used to detect these gases and offer the advantages of low power, good selectivity and temporal stability. However, EC sensors are large (1 cm3), hand-made and thus expensive ($25). Consequently, they are unsuitable for the low-cost automotive market that demands units for less than $10. One alternative technology is SnO2 or WO3 resistive gas sensors that are fabricated in volume today using screen-printed films on alumina substrates and operate at 400°C. Unfortunately, they suffer from several disadvantages: power consumption is high 200 mW; reproducibility of the sensing element is poor; and cross-sensitivity is high. © 2013 IEEE.

Detection of microcystins in environmental samples using surface plasmon resonance biosensor

An indirect inhibitive surface plasmon resonance (SPR) immunoassay was developed for the microcystins (MCs) detection. The bioconjugate of MC-LR and bovine serum albumin (BSA) was immobilized on a CM5 sensor chip. A serial premixture of MC-LR standards (or samples) and monoclonal antibody (mAb) were injected over the functional sensor surface, and the subsequent specific immunoreaction was monitored on the BIAcore 3000 biosensor and generated a signal with an increasing intensity in response to the decreasing MCs concentration. The developed SPR immunoassay has a wide quantitative range in 1-100 mu g L-1. Although not as sensitive as conventional enzyme-linked immunosorbent assay (ELISA), the SPR biosensor offered unique advantages: (I) the sensor chip could be reusable without any significant loss in its binding activity after 50 assay-regeneration cycles, (2) one single assay could be accomplished in 50 min (including 30-min preincubation and 20-min BIAcore analysis), and (3) this method did not require multiple steps. The SPR biosensor was also used to detect MCs in environmental samples, and the results compared well with those obtained by ELISA. We conclude that the SPR biosensor offers outstanding advantages for the MCs detection and may be further developed as a field-portable sensor for real-time monitoring of MCs on site in the near future. (C) 2009 Published by Elsevier B.V.

Detection and quantitative analysis of polychlorinated biphenyls in tilapia from Hawaiian waters

The purpose of this study was to investigate polychlorinated biphenyls (PCBs) contamination in tilapia (Oreochromis mossambicus) collected from the Manna stream and Ala Wai Canal of O'ahu, an island of the geographically isolated Hawaiian archipelago. Our results show that the average concentrations of PCBs varied from 51.90 to 89.42 ng g(-1) lipid weight for the sampling sites. Relative toxic potencies (RTPs) and toxic equivalencies (TEQs) were determined to be 20.38-40.60 ng TCDD g(-1) lipid weight and 2.89-4.17 ng TEQ g(-1) lipid weight by 7-ethoxy-resorufin-O-deethylase (EROD) activity analysis and calculation of PCB concentrations based on toxic equivalency factors (TEFs), respectively. Penta-chlorinated congeners were found to be predominant, which revealed that Aroclor 1254 was a possible major source of PCBs in our fish samples. PCB 118, an indicator PCBs, constituted more than 55% and 30% of the total PCBs and TEQs, respectively. In addition, PCB 118 was found to have a linear correlation to the total PCBs (R = 0.975) and TEQs (R = 0.782). Detection of concentrated PCBs in Hawaiian waters suggests a potentially adverse impact of this pollutant on human health, as well as ecological systems, and suggests the necessity of environmental monitoring and hazard assessment of PCBs within the Hawaiian Islands. (c) 2008 Published by Elsevier Ltd.

Unattended ground sensor system based on fiber optic disk accelerometer

The basic principle and critical characteristics of unattended ground sensors (UGS) based on fiber optic disk accelerometers are introduced. Mechanical principles of fiber optic disk accelerometers (FODA) and calculation methods are presented. An FODA with a high sensitivity of 120rad/g and a resonance frequency of 300Hz is designed and used for detection in military affair.