Personalized medicine is an emerging field in which clinical diagnostics information about a patient's genotype or phenotype is used to optimize his/her pharmacotherapy. This article evaluates whether planar waveguide fluorescent sensors are suitable for determining such information from patient testing in point-of-care (POC) settings. The model system was Long QT Syndrome, a congenital disease associated with single nucleotide polymorphisms (SNPs) in genes encoding for cardiac ion channels. Three different SNP assay formats were examined: DNA/DNA hybridization, DNA/PNA hybridization (PNA: "peptide nucleic acid"), and single base extension (SBEX). Although DNA/DNA hybridization produced a strong intensity-time response for both wildtype and SNP analytes in a 5-min assay at 32°C, their hybridization rates differed by only 32.7%, which was insufficient for clinical decision-making. Much better differentiation of the two rates was observed at 53°C, where the wildtype's hybridization rate was two-thirds of its maximum value, while that of the SNP was essentially zero. Such all-or-nothing resolution would be adequate for clinical decision-making; however, the elevated temperature and precise temperature control would be hard to achieve in a POC setting. Results from DNA/PNA hybridization studies were more promising. Nearly 20-fold discrimination between wildtype and SNP hybridization rates was observed in a 5-min assay at 30°C, although the low ionic strength conditions required necessitated a de-salting step between sample preparation and SNP detection. SBEX was the most promising of the three, determining the absolute identity of the suspected polymorphism in a 5-min assay at 40°C.
Oligonucleotide probes derived from (1) the T3 RNA polymerase promoter sequence (T3) and (2) prostate-specific antigen messenger RNA (PSA) were prepared and labeled with a red-emitting fluorescent dye (Cy5). The complimentary oligonucleotides were prepared and labeled with biotin. Initially, a feasibility study was performed in which the hybridization rate of the T3/anti T3 Oligonucleotide pair was examined. Specifically, biotinylated anti T3 was immobilized to a neutravidin-coated waveguide and solutions containing increasing concentrations of Cy5-labeled T3 were injected into the biosensor. Fluorescence emission was detected with an evanescent wave imaging fluorometer. The hybridization reaction proceeded rapidly with a significant amount of binding occurring during the first 5 minutes. A Michaelis-Menton kinetics model was used to analyze hybridization rate data and gave values of 78 nanomolar for the apparent affinity of the hybridization reaction and 1.4 picomolar for the analytical sensitivity of the hybridization assay. In subsequent studies the hybridization rate of the PSA/anti PSA oligonucleotide pair was examined. Biotinylated anti PSA was immobilized to the waveguide and solutions containing increasing concentration of Cy5-labeled PSA were injected into the biosensor. The hybridization rate observed for formation of the PSA/anti PSA pari was comparable to the high rates observed for the T3/anti T3 pari. Lastly, the selectivity of the biosensor was examined using an Oligonucleotide probe derived from human glandular kallikrein, which exhibits a high degree of homology to PSA. The two Oligonucleotide probes only differed in 7 out of 20 positions. Interestingly, the hybridization rate observed for Cy5-labeled hGK was very low - not statistically different from the non-specific binding rate of the hybridization assay.
An evanescent planar waveguide Mark 1.5 instrument was used to detect simulants of biological warfare agents; ovalbumin (OV), MS2 bacteriophage, BG, and Erwinia herbicola (EH). Polyclonal tracer antibodies were labeled with the fluorescent dye, Cy5. Discrete bands of polyclonal capture antibodies were immobilized to a polystyrene planar waveguide with molded integral lenses. An ST-6 CCD camera was used for detection. OV. MS2 and BG were detected in a simultaneous 3 by 3 array; with a total of nine measurements within 6 minutes. EH was analyzed in a separate array. Results were evaluate dat the US Army Joint Field Trials V, at the Dugway Proving Grounds. Over a 10 day period, 32 unknown samples were analyzed daily for each simulant. Detection limits: OV 10 ng/ml, MS2 107 pfu/ml, BG 105 cfu/ml. EH was detectable at 5 X 105 cfu/ml. Overall false positives were 3.0 percent. Therefore, the Mark 1.5 instrument, with a parallel array of detectors, evanescent flourescent excitation, and CCD imaging provides for rapid, sensitive, and specific detection of biological warfare agent simulants.
The goal of our research program is to develop an evanescent wave immunoassay system that can be used in point-of-care and critical care settings. Several key attributes are required to accomplish this goal: (1) the assay system should be at least as sensitive as present day immunoassays; (2) assay time should be 5 minutes or less; (3) the assay protocol should be relatively simple; (4) the sensor should be capable of performing more than one assay on a single specimen; (5) the assay system should be able to accommodate specimens such as serum, plasma and whole blood; and (6) the sensor should be an inexpensive, disposable cartridge. Our laboratory has developed an injection-molded planar waveguide sensor that meets most, if not all, of these attributes. This sensor has been evaluated in a number of different immunoassays for analytes such as bovine serum albumin, human chorionic gonadotrophin, creatine phosphokinase MB and cardiac troponin I.
Immunoassays based upon evanescent wave interactions are finding increased biosensing application. In these devices, the evanescent tail associated with total internal reflection of an incident beam at the substrate/solution interface provides sensitivity for surface-bound protein over bulk molecules, allowing homogeneous assays and real-time measurement of binding dynamics. Among such systems are surface plasmon resonance sensors and a resonant mirror device. Several research groups are also developing fluorescent fiberoptic or planar waveguide sensors for biomedical applications. We describe a second-generation planar waveguide fluoroimmunoassay system being developed in our laboratory which uses a molded polystyrene sensor. The 633-nm beam from a laser diode is focused into the 500 micrometer- thick planar waveguide by an integral lens. Antibodies to the desired analyte (hCG) are immobilized on the waveguide surface and fluorescence from bound analyte/tracer antibodies in a sandwich format is imaged onto the detector. The geometry of the waveguide allows several zones to be detected, providing the capability for on-sensor calibration. This sensor has shown picomolar sensitivity for the detection of hCG.
We demonstrate the use of a two-channel flowcell for fluorescent immunoassays. The flowcell contains a planar silica waveguide for evanescent excitation of the fluorophores, and the planar waveguide surface provides the solid support for immobilization of the antibodies. The detection system is composed of a grating spectrometer and a CCD camera for spectral characterization of the emitted signals. Two methods of sensing have been studied: a displacement-type technique and a sandwich-type assay. The sensitivity achieved for measuring concentrations of HCG by the sandwich method is sub-picomolar. Also, we have experimentally compared the signal strengths for two alternative ways of excitation and collection, and determine that waveguide excitation/side collection has some practical advantages over side excitation/waveguide collection.
The goal of our research program is to develop competitive and sandwich fluoroimmunoassays with high sensitivity and fast response time, that do not require external reagents. Our approach to this problem is to employ an optical immunoassay based on total internal reflection fluorescence (TIRF). Specifically, monoclonal antibodies are immobilized on a planar waveguide. Total internal reflection of light in the planar waveguide sets up an evanescent field which extends about 2000 angstroms from the interface. In the competitive immunoassay, a fluorescent label is coupled to a small synthetic antigen which is packaged with the antibody. In the absence of analyte, the fluorescently labeled antigen binds to the antibody and is excited by the evanescent field. Upon the addition of analyte, the fluorescently labeled antigen molecules are displaced by unlabeled antigen molecules and diffuse out of the evanescent field. In the sandwich assay, a primary or `capture' antibody is immobilized on the planar waveguide, and a secondary or `tracer' antibody (which is labeled with a fluorescent dye) is added to the bulk solution. In the absence of analyte, the tracer antibody remains in solution and very little fluorescence is observed. However, upon addition of analyte, a `molecular sandwich' is formed on the waveguide, composed of: (1) the capture antibody; (2) the analyte; and (3) the tracer antibody. Once this sandwich forms, the tracer antibody is within the evanescent field and fluoresces. Fluorescence emission is detected by a charged- coupled device (CCD). Using this approach, we have developed a prototype immunosensor for the detection of human chorionic gonadotropin (hCG). This device meets our design goals and exhibits a sensitivity of 0.1 - 1 pmolar.
We have analyzed and fabricated two different coupling schemes to meet the requirements for a convenient means of coupling into a planar waveguide immunosensor that is relatively insensitive to beam alignment. These are the `launch' coupler and the grating coupler. Each possesses advantages and disadvantages, depending mainly on the thickness (mode number) of the waveguide to be illuminated. For example, the launch coupler is best suited to a thick (highly multimode) waveguide and is less efficient for a thin (few mode) guide. Our experimental results verify predictions of a ray theory developed to give coupling efficiency for a variety of coupling parameters.
A key factor in the analysis of evanescently coupled optical sensors, such as the planar waveguide immunosensor analyzed here, is the efficiency of coupling between the optical waveguide modes and the fluorescent sources located on the surface of the waveguide. This is an important parameter in determining the sensor's sensitivity to the analyte. We calculate this efficiency for several different sensor configurations using the finite-difference time-domain numerical technique, and find that the efficiency of one-way coupling can vary widely depending upon the fluorescent source polarization, phase, and distance from the surface, as well as the waveguide mode number and thickness. In particular, we find that when the layer containing the fluorescent molecules is uniform in refractive index, the coupling efficiency is larger than when the local environment possesses an irregular index.
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