Development of a microfluidic device for detection of pathogens in oral samples using upconverting phosphor technology (UPT).

Confirmatory detection of diseases, such as HIV and HIV-associated pathogens in a rapid point-of-care (POC) diagnostic remains a goal for disease control, prevention, and therapy. If a sample could be analyzed onsite with a verified result, the individual could be counseled immediately and appropriate therapy initiated. Our group is focused on developing a microfluidic "lab-on-a-chip" that will simultaneously identify antigens, antibodies, RNA, and DNA using a single oral sample. The approach has been to design individual modules for each assay that uses similar components (e.g., valves, heaters, metering chambers, mixers) installed on a polycarbonate base with a common reporter system. Assay miniaturization reduces the overall analysis time, increases accuracy by simultaneously identifying multiple targets, and enhances detector sensitivity by upconverting phosphor technology (UPT). Our microfluidic approach employs four interrelated components: (1) sample acquisition-OraSure UPlink collectors that pick-up and release bacteria, soluble analytes, and viruses from an oral sample; (2) microfluidic processing-movement of microliter volumes of analyte, target analyte extraction and amplification; (3) detection of analytes using UPT particles in a lateral flow system; and (4) software for processing the results. Ultimately, the oral-based microscale diagnostic system will detect viruses and bacteria, associated pathogen antigens and nucleic acids, and antibodies to these pathogens.

Rapid assay format for multiplex detection of humoral immune responses to infectious disease pathogens (HIV, HCV, and TB).

A novel assay is described for multiplex detection of antibodies against different pathogens from a single sample. The assay employs a modified lateral flow format (consecutive flow, CF) together with a sensitive reporter particle technology (up-converting phosphor technology, UPT) that allows for fully instrumented assay analysis. Lateral flow (LF) strips developed for the detection of human antibodies against human immunodeficiency virus type-1 and -2 (HIV-1 and -2) with additional capture zones to detect antibodies against Myobacterium tuberculosis (TB) and hepatitis C Virus (HCV) provided the strips to test multiplexing. Data are presented that show the performance of the TB and HCV test, as well as two multiplex assays, TB with HIV and HCV with HIV. The TB/HCV assays demonstrate excellent detection capability, and HIV multiplexing does not affect the qualitative test result. The bench-top CF format was converted to a microfluidic platform and a first prototype semiautomated chip capable of performing CF is presented here.

Evaluation of UPlink-RSV: prototype rapid antigen test for detection of respiratory syncytial virus infection.

A prototype rapid antigen test for the on-site detection of respiratory syncytial virus (RSV) infection was developed and evaluated. The platform uses instrumented assay analysis, eliminating potential operator bias in the interpretation of the test result that may occur with visually interpreted rapid antigen assays. The device was tested as the first point-of-care (POC) infectious disease application of novel reporter up-converting phosphor technology (UPT) using a specifically designed portable UPT reader (UPlink). Assays were performed by mixing nasopharyngeal specimen with RSV-specific UPT reporter particles and addition of the mixture to a disposable cassette containing a lateral flow (LF) strip with RSV capture antibodies. UPT reporters bound on the specific capture zone were analyzed with the UPlink reader. Reproducibility testing of the UPlink-RSV (UPR) test by naïve users confirmed the potential of UPlink for POC applications where testing is not always performed by highly trained medical staff. The performance of UPR was further evaluated with clinical nasopharyngeal specimens. A prospective study at an independent test site demonstrated clinical parameters of 90% sensitivity and 98.3% specificity with an overall correlation of 96.2% as compared to viral culture with RT-PCR verification. These results are in agreement with in-house retrospective studies and results obtained with other available commercial rapid antigen assays.

An amplification-free hybridization-based DNA assay to detect Streptococcus pneumoniae utilizing the up-converting phosphor technology.

OBJECTIVES: Development of a generally applicable sensitive hybridization-based assay devoid of any target amplification for the detection and identification of (pathogenic) bacterial and viral species. DESIGN AND METHODS: Using a sandwich hybridization format, the presence of a species-specific nucleic acid sequence is detected by means of Lateral Flow (LF) and Up-converting Phosphor Technology (UPT, a luminescent tracer). As a model, detection of the pathogen Streptococcus pneumoniae was investigated using a probe against the single-copy lytA gene. RESULTS: Detection of S. pneumoniae (in particular a 1200 p lytA sequences) required less than 1 ng of genomic DNA (approximate size 2.2 Mb). Hybridization and detection were performed in a complex background containing 10 microg fish sperm DNA. CONCLUSIONS: The results indicate the possibility to detect nucleic acid targets in nonamplified DNA samples using easy, inexpensive, amplification-free hybridization-based assays and the ultra sensitive UPT reporters. Employment of UPT allows to by-pass target amplification and therefore brings genetic-based testing a step closer to the point-of-care environment. Detection of S. pneumoniae with only 1 ng of DNA indicates a potential for applications in the field of infectious diseases.

Lateral-flow and up-converting phosphor reporters to detect single-stranded nucleic acids in a sandwich-hybridization assay.

Up-converting Phosphor Technology (UPT) particles were used as reporters in lateral-flow (LF) assays to detect single-stranded nucleic acids. The 400-nm phosphor particles exhibit strong visible luminescence upon excitation with infrared (IR) light resulting in the total absence of background autofluorescence from other biological compounds. A sandwich-type hybridization assay was applied using two sequence-specific oligonucleotides. One of the oligonucleotides probes was covalently bound to the UPT particle (reporter) for direct labeling and detection, whereas the second oligonucleotide probe contained biotin for capture by avidin during LF. The whole procedure of hybridization, UPT-LF detection, and analysis required a minimum time of 20 min. Moreover, aiming at minimal equipment demands, the hybridization conditions were chosen such that the entire assay could be performed at ambient temperature. During lateral flow, only targets hybridized to both capture and detection oligonucleotide were trapped and detected at an avidin capture line on the LF strip. Analysis (IR scanning) of the strips was performed in an adapted microtiter plate reader provided with a 980-nm IR laser for excitation of the phosphor particles (a portable reader was also available). Visible luminescence was measured and presented as relative fluorescence units (RFU) allowing convenient quantitation of the phosphor signal. With the assay described here as little as 0.1 fmol of a specific single-stranded nucleic acid target was detected in a background of 10 microg fish sperm DNA.