BioMedomics Point of Care Diagnostic Platforms: Lateral Flow Immunoassay, and Time Resolved Fluorescence Detection

Most popular Point of Care (POC) platforms are lateral-flow tests and electrochemical enzymatic tests. BioMedomics has developed a new POC platform based on the principle of lateral flow technologies and time-resolved detection. POC Platforms: Lateral flow tests, or immunochromatographic strips, were first introduced as qualitative urine pregnancy tests used in doctors offices and at home. They have evolved into rapid tests for a range of analytes, including HIV, respiratory diseases, drugs-of-abuse, cardiac markers, and infectious diseases. Lateral flow tests are a form of immunoassay in which the test sample flows along a solid substrate via capillary action. After the sample is applied to the test, it encounters a colored reagent, which mixes with the sample and transits the substrate encountering lines or zones which have been pretreated with an antibody or antigen. Depending upon the analytes present in the sample the colored reagent can become bound at the test line or zone. Lateral flow tests can operate as either competitive or sandwich assays. Although their use has become widespread, lateral-flow tests have sensitivity limitations and tend to be semiquantitative. Recent improvements in lateral-flow testing have been made using both fluorescent and magnetic labels. Immunoassays are tests that take advantage of the specific binding of an antibody to its antigen. See Figure 1. The main characteristic of immunological techniques is the appropriate labeling of the antibody or the antigen. This label helps create a signal that correlates with the immunoreaction and allows the detection of the analyte of interest. In laboratory assays, various wash steps are required to remove free labeled or unlabeled reactants and allow the detection of the analyte by the bound and labeled reactants. Test results Figure 1: Lateral Flow Immunoassay Principle are then compared with a calibration curve to quantify them. In contrast, well-known lateral-flow immunoassays (LFIA), such as pregnancy

tests, do not require the washing steps. In a lateral-flow test system, the antibody-antigen reaction, as well as the removal of excess reactants, takes place by chromatographic separation. The detector reagent (i.e., the labeled antibody) and the sample wick through the pads or membranes of the test strip. At a capture line, the detector reagent interacts with the capture reagent, which has been immobilized on the membrane. The test result is then evaluated visually, typically by two lines a test line and a control line. Electrochemical enzymatic tests were developed primarily for measuring the glucose levels of diabetic patients. This technology has also evolved, and electrochemical tests are available for coagulation and immunoassays, such as the i-Stat test by Abbott Point-of-Care (East Windsor, NJ) for troponin I. The next generation of POC devices will need to meet a number of key requirements (see Table 1). Some requirements are more critical than others and will vary depending on the analyte, such as detection limit, Table 1: Lateral Flow Immunoassay, Key Requirements dynamic range, and precision. However, in order to use a finger-stick blood sample in POC tests, the sample volume requirements should be minimized and the test should be conducted with whole blood. If a single technology could fulfill all of the requirements in the Table, it could be used in a range of diagnostic applications. New Time-Resolved Lateral Flow Assay System: BioMedomics is also developing a new POCT platform using advanced patented timeresolved lateral flow assay technologies. Compared with other immunoassay formats, the major advantages of LFIA are their ease of use; aside from the dispensing of the sample, no additional handling steps are usually necessary. Thus, LFIA can be easily performed by non-trained users and used on-site during sample collection. The simplicity of the tests paired with their quick return of results (215 minutes), means that testing is cost-effective. LFIA represent an appropriate POC and field-use technology that can be brought to market quickly for a small investment, and can be applied to a broad range of applications. Despite these advantages, LFIA are often limited to screening applications. This is because LFIA, in their present form, are not easily quantifiable and are not sensitive enough for certain applications. Recently a new technology has been developed utilizing fluorescence detection assays for the LFIAs. This fluorescence based assay technology improves sensitive and quantitative clinical diagnosis. However, because of the detection background from clinical samples and assay strip membrane materials, the sensitivity is not good enough for critical biomarker assays.

In response to the problems with conventional fluorescence detection techniques, we will apply a "time-resolved" fluorescence detection technique. As shown in Figure 2, TimeResolved Fluorescence involves exciting the fluorescent label with a short pulse of light, then waiting a certain time (e.g., between approximately 100 to 200 microseconds) after excitation before measuring the remaining long-lived fluorescent signal. In this manner, any short-lived fluorescent background signals and scattered excitation radiation are eliminated.

Figure 2: Principle of Time-Resolved Fluorescence Detection for LFIA

Compared with other detection platforms, Time-Resolved Fluorescence detection has many advantages as shown in Table 2. It is highly sensitive, specific and quantitative which is very suitable for POC applications.

Table 2: Advantages of Time-Resolved Fluorescence Detection

BioMedomics is developing new time-resolved fluorescent labeling techniques and POC portable fluorescence detection platforms for LFIA-based biomedical applications. The existing prototype of the Time-Resolved Fluorescence Detection LFIA is shown in Figure 3. In this platform, electronics, optical and mechanical components have been integrated and tested.

One of the key technology development related to Time-Resolved Fluorescence LFIA technology is the long lifetime fluorescent particle probe development. BioMedomics has licensed the technology related to this novel probe fabrication process. The initial long lifetime probe work was successful as shown in Figure 4. The initial time-resolved POC detection was carried out and compared with standard conventional fluorescence detection in POC platform. In the prototype testing, the TimeFigure 3: Time-Resolved Fluorescence LFIA device. Resolved LFIA detection limitation is at <0.2 pg/ml as shown in Figure 5. This detection sensitivity is significantly higher than any other existing POC platforms. With novel fluorescence labeling methods and unique detection method, the Time-Resolved POC platform can significantly increase detection sensitivity by orders of magnitude compared with traditional lateral-flow labeling and detection methods.

Figure 4: Photos of long lifetime fluorescent probe particles used for Time-Resolved LFIA

Figure 5: Preliminary testing results with Time-Resolved LFIA

The new technologies associated with BioMedomics development will open many new and broad business opportunities while still being able to detect results with a simple, fast, reliable, and portable platform. With the introduction of these new groundbreaking technologies, BioMedomics business will have a great impact on POCT and Personalized Medicine diagnostics.