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A multidisciplinary group of scientists from the University of Pennsylvania joined with investigators from the dominant company in salivary diagnostics, OraSure Technologies Inc, and Leiden University Medical College to collaborate on this project. The technology platform being developed is focused on oral diagnostics to detect viral and bacterial pathogens, using HIV-1 and B. cereus as prototypes. The involvement of OraSure technologies, Inc., should accelerate the commercial development of this technology, either directly via OraSure or alternatively by a larger corporate partnership. It is important to note that this same technology platform is applicable to analyses of other types of samples (blood, urine, CSF, tears, nasal swabs, hard surface swabs). Furthermore, the technology can be applied to the diagnosis of a wide variety of bacterial and viral targets including agents identified by the CDC as likely pathogens for bioterrorism, such as anthrax and smallpox virus (Variola). While clinical laboratories have developed tests to diagnos many bacterial and/or viral pathogens, few are multiplexed and none are applicable for point-of-care applications.

Sample Collection

The UPlink™ collector was selected in year 2 for development in this project (Appendix Table 1). This collector picked up and released buffer, whole saliva, amylase, and bacteria and was compatible with PCR (Appendix Figure 1). In addition, the device is already approved for human use and can be easily modified to increase sample capacity, should that prove necessary to increase detection sensitivity (Appendix Figure 2).

Assay Development

A number of parameters associated with both the PCR assays and lateral flow (LF) detection have been evaluated, and each step of the process has been optimized for maximum sensitivity and compatibility with a microfluidic platform.

PCR Detection of B. cereus and HIV

We determined the optimum primer concentration, DNA concentration, buffer composition, BSA concentration, time and temperature optima for primer sets that produce amplicons of 700, 305, and 105 bp from genomic B. cereus DNA. The data obtained with benchtop and real-time PCR protocols were the basis for the design and optimization of the microfluidic PCR system

In year 3, the simultaneous amplification of nucleic acid from HIV (RNA) and B. cereus (DNA) has been demonstrated starting with a mixture of intact HIV and B. cereus (Figure 1). This experiment shows that the reverse transcription step to form HIV cDNA does not interfere with the PCR amplification of B. cereus DNA.

Lateral Flow Detection Optimization

We deconstructed the steps associated with detection of the PCR product using UPT particles to enhance signal detection. The most significant of these steps are the addition of BSA and heating or drying of the LF nitrocellulose membrane prior to scanning. Appendix Table 2 summarzes the results of the optimization parameters. A rapid RT-PCR based detection of both bacterial DNA and HIV-1 RNA utilizing UPT and a lateral flow (LF) detection platform appears feasible.

HIV antibody assay development:
the consecutive flow format

Assay Description: When compared to the FDA approved OraQuick gold assay for antibodies to HIV-1, UPT detection demonstrated higher sensitivity with a dilution series of HIV-1 positive sera. In order to use UPT-reporters in the HIV-1 assay, a consecutive flow format was utilized (Figure 2). In this format a serum sample is diluted in bufffer and applied to the lateral flow strip before the UPT-reporter is added. This allows the binding and enrichment of the small population of HIV-1 specific IgG molecules at the test line. Other IgG molecules flow past the test line and bind to the flow-control line (containing an anti-human IgG), excess antibody flows to the absorbent pad. The second step in this consecutive flow format is a wash followed by a flow of an UPTprotA reporter-conjugate. The interaction of protein A with any IgG molecule results in a signal at the test-line whenever specific HIV-1 IgG is bound. The consecutive flow system requires user training because of the multiple manipulations required and may not be applicable for an over-the-counter test system. However, when incorporated into a microfluidic device, the multiple flow steps will be automated. The full potential of the consecutive flow format and a generic reporter will be exploited with multiple test or capture lines to detect host antibodies against different infections, e.g., a combination of HIV and Hepatitis C.

Sensitivity Comparison: OraQuick HIV-1 gold and the UPT HIV-1 consecutive flow sensitivity were compared analyzing a dilution series of HIV-1 positive sera (Appendix Table 3). Note that UPT detection was three orders of magnitude more sensitive than gold. The UPT HIV-1 consecutive flow was verified with 50 HIV negative and 50 HIV positive serum samples. UPT demonstrated higher resolution as compared to gold in 2 seroconversion panels (Appendix Table 4).

HIV antibody assay development: adapting consecutive flow format to a microfluidic chip

A bench-top protocol detecting HIV-1 antibodies in plasma using UPT detection was established. The lateral flow assay includes a test-zone of HIV synthetic peptides. Detection of the HIV antibodies is then performed with an IgG generic UPT-label in a consecutive flow format. In a study using 51 HIV-1 positive plasma specimens, UPT-LF (Lateral low) scored all samples well above the control threshold determined from 50 HIV-1 negative plasma samples. Results were in concordance with the results obtained using the OraQuick® HIV-1 Gold test. Figure 3 shows that the fluorescence ratio values for all positive specimens are well separated from the negative specimens. The sensitivity of the UPT-LF assay was determined analyzing a dilution series of one of the high positive specimens. Utilization of UPT detection generates a signal well above the negative control level for a 10,000 fold dilution of an HIV-1 positive sample.

The bench-top UPT-LF protocol was then implemented in a microfluidic chip constructed of bonded layers of polycarbonate. The device utilizes thermo-pneumatic sample introduction, distribution, metering and mixing. Mixing of reagents with sample is enhanced by repeated reciprocal movement of fluid between two chambers (Fig. 4). Phase-change valves control flow which freeze the working liquid to a solid state to block the flow passage. Detection is accomplished by measuring UPT reporter particles which bound to the target zone on the nitrocellulose strip. The UPT particles are excited with an infrared laser (980 nm) and the emitted visible light (550 nm) is recorded. Preliminary tests of the microfluidic device were performed with a Digitonin/Biotin labeled DNA amplicon, UPT-Avidin lyospheres and LF strips with an anti-Digitonin test line. The results (Figure 5) indicate full compatibility and applicability of the UPT-LF format with a microfluidic chip format.