Surround optical-fiber immunoassay (SOFIA) is an ultrasensitive, in vitro diagnostic platform incorporating a surround optical-fiber assembly that captures fluorescence emissions from an entire sample. The technology's defining characteristics are its extremely high sensitivity and dynamic range, and extremely low limit of detection. SOFIA's sensitivity is measured at the attogram level (10−18 g), making it about one billion times more sensitive than conventional diagnostic techniques. Based on its enhanced dynamic range, SOFIA is able to discriminate levels of analyte in a sample over 10 orders of magnitude, facilitating accurate titering.
As a diagnostic platform, SOFIA has a broad range of applications. Several studies have already demonstrated SOFIA's unprecedented ability to detect naturally occurring prions in the blood and urine of disease carriers. This is expected to lead to the first reliable ante mortem screening test for vCJD, BSE, scrapie, CWD, and other transmissible spongiform encephalopathies. Given the technology's extreme sensitivity, additional unique applications are anticipated, including in vitro tests for other neurodegenerative diseases, such as Alzheimer's and Parkinson's disease.
SOFIA was developed as a result of a joint-collaborative research project between Los Alamos National Laboratory and State University of New York, and was supported by the Department of Defense's National Prion Research Program.
Background The conventional method of performing laser-induced fluorescence, as well as other types of spectroscopic measurements, such as infrared, ultraviolet-visible spectroscopy, phosphorescence, etc., is to use a small transparent laboratory vessel, a cuvette, to contain the sample to be analyzed.
To perform a measurement, the cuvette is filled with the liquid to be investigated and then illuminated with a laser focused through one of the cuvette's faces. A lens is placed in line with one of the faces of the cuvette located at 90° from the input window to collect the laser-induced fluorescent light. Only a small volume of the cuvette is actually illuminated by the laser and produces a detectable spectroscopic emission. The output signal is significantly reduced because the lens picks up only about 10% of the spectroscopic emission due to solid angle considerations. This technique has been used for at least 75 years; even before the laser existed, when conventional light sources were used to excite the fluorescence.
SOFIA solves the problem of low collection efficiency, as it collects nearly all of the fluorescent light produced from the sample being analyzed, increasing the amount of fluorescence signal by around a factor of 10 over conventional apparatus.
Technological advantages SOFIA is an apparatus and method for improved optical geometry for enhancement of spectroscopic detection of analytes in a sample. The invention has already demonstrated its proof-of-concept functionality as an apparatus and method for ultrasensitive detection of prions and other low-level analytes. SOFIA combines the specificity inherent in monoclonal antibodies for antigen capture with the sensitivity of surround optical detection technology. To detect extremely low signal levels, a low-noise, photovoltaic diode is used as the detector for the system. SOFIA uses a laser to illuminate a microcapillary tube holding the sample. Then, the light collected from the sample is directed to transfer optics from optical fibers. Next, the light is optically filtered for detection, which is performed as a current measurement amplified against noise by a digital signal processing lock-in amplified. The results are displayed on a computer and software designed for data acquisition.
The advantages of such a detection array are numerous. Primarily, it permits the use of very small samples at low concentration to be optimally interrogated using the laser-induced fluorescence technique. This fiber-based detection system is adaptable to existing short-pulsed detection hardware that was originally developed for sequencing single DNA molecules. The geometry is also amenable to deployment for short-pulse laser, single-molecule detection schemes. The multiport geometry of the system allows efficient electronic processing of the signals from each arm of the device. Finally, and perhaps most importantly, fiberoptic cables are essentially 100% efficient in optical transmission, having an attenuation less than 10 dB/km. Thus, once deployed for use in a facility, the fluorescence information can be fiberoptically transmitted to a remote location, where data processing and analysis can be performed.
Components of SOFIA SOFIA comprises a multiwell plate sample container, an automated means for successively transporting samples from the multiwell plate sample container to a transparent capillary contained within a sample holder, an excitation source in optical communication with the sample, wherein radiation from the excitation source is directed along the length of the capillary, and wherein the radiation induces a signal which is emitted from the sample, and, at least one linear array.
Steps in SOFIA
Assay preparation After amplifying and then concentrating the target analyte, the samples are labeled with a fluorescent dye using an antibody for specificity and then finally loaded into a microcapillary tube. This tube is placed in a specially constructed apparatus so it is totally surrounded by optical fibers to capture all light emitted once the dye is excited using a laser.
Instrumentation processing This equipment is a spectroscopic (light gathering) apparatus and corresponding method for rapidly detecting and analyzing analytes in a sample. The sample is irradiated by an excitation source in optical communication with the sample. The excitation source may include, but is not limited to, a laser, a flash lamp, an arc lamp, a light-emitting diode, or the like.
Figure 1 depicts the current version of the SOFIA system. Four linear arrays (101) extend from a sample holder (102), which houses an elongated, transparent sample container which is open at both ends, to an end port (103). The distal end of the endport (104) is inserted into an end port assembly (200). The linear arrays (101) comprise a plurality of optical fibers having a first end and a second end, the plurality of optical fibers optionally surrounded by a protective and/or insulating sheath. The optical fibers are linearly arranged, meaning that they are substantially coplanar with respect to one another so as to form an elongated row of fibers.
Applications The analyte of interest may be biological or chemical in nature, and by way of example, only may include chemical moieties (toxins, metabolites, drugs and drug residues), peptides, proteins, cellular components, viruses, and combinations thereof. The analyte of interest may be in either a fluid or a supporting medium, such as a gel.
SOFIA has demonstrated its potential as a device with a wide range of applications. These include clinical applications, such as detecting diseases, discovering predispositions to pathologies, establishing a diagnosis and tracking the effectiveness of prescribed treatments, and nonclinical applications, such as preventing the entry of toxins and other pathogenic agents into products intended for human consumption: Clinical applications – SOFIA may be used to conduct both qualitative tests (either positive or negative results) to detect or identify bacteria or viruses, and quantitative tests (measuring substances) to detect or quantify biological constants or markers, which are substances produced by the body in the presence of, for example, an infectious disease (to allow determination of viral load, for instance, in AIDS therapy, or the level of toxicity in drugs of abuse detection). Nonclinical applications - As an immunoassay, SOFIA can potentially be used on a wider scale to monitor the quality of food, pharmaceuticals, cosmetics, or water, as well as general environmental parameters and agricultural products. The ability to detect and screen bacteria and toxins for a wide range of products is a growing and more complex requirement as may be evidenced by the increase incidence of food- and animal-borne diseases, such as E. coli, Salmonella, BSE, avian influenza, etc.
Ante mortem test for prion diseases SOFIA has been used to rapidly detect the abnormal form of the prion protein (PrPSc) in samples of bodily fluids, such as blood or urine. PrPSc is the marker protein used in diagnostics for transmissible spongiform encephalopathies (TSEs), examples of which include bovine spongiform encephalopathy in cattle (i.e. “mad cow” disease), scrapie in sheep, and Creutzfeldt–Jakob disease in humans. Currently, no rapid means exists for the ante mortem detection of PrPSc in the dilute quantities in which it usually appears in bodily fluids. SOFIA has the advantages of requiring little sample preparation, and allowing for electronic diagnostic equipment to be placed outside the containment area.
Background TSEs, or prion diseases, are infectious neurodegenerative diseases of mammals that include bovine spongiform encephalopathy, chronic wasting disease of deer and elk, scrapie in sheep, and Creutzfeldt–Jakob disease (CJD) in humans. TSEs may be passed from host to host by ingestion of infected tissues or blood transfusions. Clinical symptoms of TSEs include loss of movement and coordination and dementia in humans. They have incubation periods of months to years, but after the appearance of clinical signs, they progress rapidly, are untreatable and invariably are fatal. Attempts at TSE risk-reduction have led to significant changes in the production and trade of agricultural goods, medicines, cosmetics, blood and tissue donations, and biotechnology products. Post mortem neuropathological examination of brain tissue from an animal or human has remained the ‘gold standard’ of TSE diagnosis and is very specific, but not as sensitive as other techniques.
To improve food safety, it would be beneficial to screen all the animals for prion diseases using ante mortem, preclinical testing, i.e., testing prior to presentation of symptoms. However, PrPSc levels are very low in presymptomatic hosts. In addition, PrPScs are generally unevenly distributed in body tissues, with highest concentration consistently found in nervous system tissues and very low concentrations in easily accessible body fluids such as blood or urine. Therefore, any such test would be required to detect extremely small amounts of PrP and would have to differentiate PrPC and PrPSc.
Current PrPSc detection methods are time-consuming and employ post mortem analysis after suspicious animals manifest one or more symptoms of the disease. Current diagnostic methods are based mainly on detection of physiochemical differences between PrPC and PrPSc which, to date, are the only reliable markers for TSEs. For example, the most widely used diagnostic tests exploit the relative protease resistance of PrPSc in brain samples to discriminate between PrPC and PrPSc, in combination with antibody-based detection of the PK-resistant portion of PrPSc. It has as yet not been possible to detect prion diseases by using conventional methods, such as polymerase chain reaction, serology, or cell culture assays. An agent-specific nucleic acid has not yet been identified, and the infected host does not elicit an antibody response.