Dynamic Passivation with BSA Overcomes LTCC Mediated Inhibition of PCR

The increasing use of low temperature co-fired ceramic (LTCC) for the fabrication of biological microfluidic devices necessitates further research on LTCC biocompatibility. In this study we explore the inhibitory effect of DuPont\u27s 951 LTCC on Polymerase Chain Reaction (PCR), and demonstrate a novel mechanism to increase biocompatibility between LTCC and PCR with the addition of a common passivation substance, bovine serum albumin (BSA). We show that DuPont\u27s 951 LTCC binds negatively charged proteins including BSA and ovalbumin (OVA). This is a significant discovery as proteins (enzymes) are an essential component of most biological reactions, and a frequent addition to microfluidic devices. A proposed model for LTCC inhibition of PCR by enzyme adsorption is presented

Bio-Terror Agent Multiplex Detection Compatible with Low Temperature Co-Fired Ceramic

New methods of bioterror detection are being developed from novel materials that simultaneously detect several biological threat gents. The recently developed material, Low Temperature Co-fired Ceramic (LTCC), is in many ways ideal for biosensors involved in bioterror detection. It is resistant to corrosion, allows co-manufacture with electric components, has a similar expansion coefficient to co-fired circuitry materials, can be autoclaved or baked, and is inexpensive to manufacture. However, the material has been known to inhibit some enzymatic reactions (through an undefined mechanism), including the most common DNA amplification enzyme Taq polymerase. Our laboratory is part of a team that is developing the first generation of biosensors made from LTCC. This study characterizes the effects of several possible causes of Taq polymerase inhibition, and demonstrates that the inhibition can be overcome by the addition of negatively charged proteins, which competively bind LTCC. Also described here is the first multiplex PCR assay that simultaneously detects very low levels of target sequences from three select bioterrorism agents in the presence of LTCC. The targets detected in this multiplex assay are from the Category A select agents B. anthracis, F. tularensis, and Y. pestis. This new assay fulfills the two key requirements of bioterror detection: it is both sensitive and selective, reducing the risk of false negative and false positive results respectively. Three design features optimize correct analysis in this multiplex reaction. First, two gene targets are amplified from each of the three organisms, enhancing the sensitivity of the assay, and decreasing the likelihood of obtaining a false positive or false negative result. Second, the majority of the PCR targets are located on plasmids, which generally occur in higher copy numbers than chromosomal DNA, further increasing the sensitivity of the assay. Third, most of the gene targets are virulence factors, which can be used to discriminate bioterror agents from non-virulent close relatives. Simultaneous amplification from five of the six PCR targets was confirmed in the presence of extraneous environmental DNA, at a sensitivity of ten or fewer target molecules per reaction. One target could not be assayed due to bio-safety level restrictions on the Pasteur strain of B. anthracis. A proposed model for LTCC inhibition of PCR is presented in the discussion. It is hypothesized that LTCC binds negatively charged proteins like DNA polymerase (the enzyme responsible for PCR amplification) impairing or preventing catalysis. In support of this model, LTCC is shown to bind the negatively charged protein ovalbumin. Furthermore, another negatively charged protein, bovine serum albumin (BSA), is shown to competitively bind to LTCC freeing ovalbumin; BSA addition also alleviates PCR inhibition by LTCC. In further support of the proposed model, the addition of negative chloride ions is also shown to overcome PCR inhibition, possibly though direct interaction with the putatively charged LTCC surface

A Prototype Continuous Flow Polymerase Chain Reaction LTCC Device

There is a growing need for remote biological sensing in both laboratory and harsh field environments. Sensing and detection of biological entities such as anthrax, Ebola and other micro-organisms of interest involves sampling of the environment, amplification, analysis and identification of the target DNA. A key component of such a sensor is a low cost, portable, reusable, continuous flow polymerase chain reaction (PCR) thermal cycler. Fabrication with low temperature co-fired ceramics (LTCC) can provide a reusable low cost device capable of operating in a wide range of environments. The design and manufacture of a prototype continuous flow micro-fluidic PCR device using low temperature co-fired ceramic is presented. Initial modeling of flow characteristics and heat transfer was carried out in SolidWorks™. The prototype device employs resistance heaters below the channels, buried and surface thermocouples for temperature monitoring, and air gaps for thermal isolation