ASR&D Biosensor Development
ASR&D is actively developing tools for point-of-care (POC) diagnosis of infectious agents based on the company's patented acoustic wave array affinity biosensor technology. This goal of this work is a diagnostic system consisting of a reusable hand-held reader capable of simple push-button operation for automated analysis of samples, and cost-effective disposable microfluidic sensor cartridges that have been functionalized to provide optimal identification of multiple clinically relevant target infectious agents. ASR&D's biosensor chip incorporates microfluidic channels and nanostructured biologically specific binding films to provide rapid, simultaneous, direct detection of multiple infectious agents. The results of each test will be provided locally by the electronic reader in under 20 minutes, and can be reported remotely through a variety of wireless options to centralized reporting facilities. The cost effective nature of these disposable biosensor cartridges and electronic readers, combined with their ease of use by untrained personnel, will enable widespread testing for infections and monitoring of outbreaks, even in resource limited settings.
The biosensor chip being developed by ASR&D is a platform technology that can be modified to detect a wide range of infectious agents, including bacteria and other cells, viruses, proteins, nucleic acids, and cancer markers, and can also be used to detect small molecules such as hormones, drugs, and pesticides in liquids. As such, it has numerous potential applications to POC testing for human and veterinary medicine. Sexually transmitted diseases (STDs) pose a major public health problem worldwide, as indicated by the World Health Organization, which states: "Sexually transmitted infections are a major global cause of acute illness, infertility, long term disability and death, with severe medical and psychological consequences for millions of men, women, and infants" [1]. Because of this clear unmet need and the potential of ASR&D's cost-effective biosensor technology to address this need, the company's initial product development has focused on the accurate, specific, real-time diagnosis of STDs such as Chlamydia trachomatis and other STDs known to co-exist with C. trachomatis in a statistically significant manner in symptomatic and asymptomatic patients.
The technology being developed is somewhat related to existing piezoelectric affinity biosensors, but provides substantially higher sensitivity than quartz crystal microbalance (QCM) devices. The enhanced sensitivity sensor arrays utilize thinned single crystal piezoelectric substrates that propagate layer guided shear horizontal acoustic plate mode (LG-SH-APM) waves in sensing regions, and incorporate multiple on-chip microfluidic channels with individual nanostructured biologically specific coatings to provide simultaneous direct identification of multiple target analytes.
These biosensor arrays are being developed with volume manufacturing in mind, and overcome the problems with complex packaging and fluid handling that have been an impediment to the widespread commercial use of existing SAW-based biosensors. ASR&D's biosensor arrays eliminate the requirement for complex packaging and fluid handling by incorporating multiple microfluidic channels on-chip, which provide both thinned crystal membranes for LG-SH-APM propagation, and fluid propagation paths for bioreceptor coating during manufacture and sample distribution for multiplexed testing during use. This novel device structure will allow mass production and utilization of standard wafer processing and device packaging techniques, which can be combined with low-cost plastic microfluidic cartridges for sample handling and sensor/reader interface to enable low cost, high volume disposable sensor cartridge production.
Figures 1 and 2 below show schematics of ASR&D's multichannel biosensor chip as viewed from the electrical (Figure 1) and fluidic (Figure 2) sides. A conventional 0.35-0.5 mm thick LiNbO3 wafer, polished on both sides, is used as the piezoelectric substrate. The crystal has (in this example) five microfluidic channels etched in the fluidic side of the chip. A side view of these channels is shown in the upper portion of Figure 2, and a top view of the channels is shown in the lower portion of Figure 2. As shown in Figure 1, each channel has multiple metal electrode structures on the opposite surface (the electrical side of the chip), each designed to launch, receive, and/or reflect the acoustic wave. In this example, different frequency devices are shown on the different channels, in a differential delay line configuration. Alternate embodiments of this biosensor utilize resonant structures, coded transducer and/or reflector structures, and other SAW elements, illustrating the large number of degrees of freedom available to the designer. This device provides flexibility that allows the designer to utilize the frequency and/or device structure that is optimal to measure the biological target in each channel separately. Thus, if one channel requires nanoparticle bioreceptors for direct detection, while another channel uses antibodies in a sandwich assay, the acoustic characteristics of each of these channels can be separately modified to measure binding to each of these bioreceptor layers optimally. Although the five channels are shown as equal in width, the channel geometry is yet another added degree of freedom available to the designer. Again considering Figure 2, the colored regions represent the specific bioreceptor layers that have been deposited on the channel fluid surface (opposite the electrical surface on which the acoustic generation and detection elements are made).
Figure 1. Array biosensor with five channels, electrical side (bottom) and cross section (top).
Figure 2. Array biosensor with five channels, fluidic side (bottom) and cross section (top).
Figure 3 shows a side view of the array chip of Figures 1 and 2, capped with a compliant cover (shown in light blue) that seals to the top of the walls between and around the channels, and also provides fluid connections to off-chip instrumentation. As shown in Figure 3, during chip manufacture, this cover will be connected to multiple fluid sources in order to functionalize each channel with the specific bioreceptors needed to detect the target analyte of interest in that channel. While Figure 3 illustrates functionalization of a single device that is already flip-chip mounted in a standard surface mount package, processes are also being evaluated for us in wafer scale manufacturing, utilizing standard dimensions for automated micro-pipette distribution of the required solutions.
Figure 3. Each channel can be separately coated with selected nanostructured bioreceptor films
Figure 4 shows additional detail on the microfluidic encapsulation to be used for the sensor arrays. As shown in the figure, the manufacturing cap can be made to have different fluid connections at the two ends of the channels, allowing introduction of functionalization solutions during manufacturing, with waste fluids exhausted through a common plenum. After channel functionalization is complete, a buffer solution will be introduced to the channels for chip encapsulation prior to dicing, packaging in the fluidic cartridge, shipping, and eventual use. The die will be flip-chip mounted in a low-cost fluidic cartridge, which will most likely be a composite of low temperature co-fired ceramic (LTCC) and thermoplastic construction, with compliant materials used to seal the die to the sides of the package for added mechanical stability and to ensure no liquid leakage to the area beneath the die (with the electrical connections) occurs during sample introduction. For use, the microfluidic cartridge encapsulating the sensor will introduce the sample through the common plenum, which distributes the sample to the parallel channels, with waste flowing out through the separate connectors at the opposite end of the device. This approach allows a permanent, wafer level cap to be installed during manufacturing, and avoids the requirement to remove and replace this cap with a new cover for device shipping, storage, and use.
Figure 4. Fluidic encapsulation for manufacturing and sample introduction
[1] World Health Organization. Global prevalence and incidence of selected curable sexually transmitted infections: overview and estimates. Geneva (Switzerland). Available at: http://www.who.int/hiv/pub/sti/