Diffractive Optics Technology: A Novel Detection Technology for Immunoassays

Abstract

There is an increasing need for high-sensitivity immunoassays that can be used in point-of-care patient testing of complex media. For example, analytes such as the natriuretic peptides and recently discovered sepsis markers are found in blood in very low picomolar concentrations (1, 2 ). Although advances have been made in the use of fluorescent, chemiluminescent, and other labels to measure markers at lower detection limits, background interference from biological samples and detection instrumentation remains problematic. Optical biosensors offer the promise of label-free real-time measurements, but their application to quantification of analytes in complex media is impaired by higher detection limits and is susceptible to changes in refractive indices or nonspecific surface binding. Moreover, the costliness of these devices has largely prohibited bedside implementation. In this report, we detail the use of a novel diffractive optics technology (dot™) that takes advantage of the inherent properties of diffractive optics to deliver a cost-effective, portable, robust, optical biosensor that detects analytes at picomolar concentrations in complex media.In the dotLab TM System, coherent light striking a nonrandom pattern of capture molecules on the dotLab Sensor creates constructive and destructive interferences that produce a well-defined diffraction image. As molecules bind to the capture molecules, the height of the diffraction pattern is increased, which in turn increases the diffraction efficiency and the diffractive order intensity. A photodiode monitors the intensity of the diffractive order, which is correlated to analyte concentrations. Because diffraction is inherently self-referencing, the transduction of binding events is dependent on the initial pattern, and an increase in diffractive order intensity will occur only if molecules bind exclusively to the patterned capture reagents. Therefore, nonspecific binding to both the patterned and nonpatterned regions will not affect the signal, a characteristic that offers an important advantage over other optical biosensor systems in which any surfacebinding event will cause an increase in signal.Previous diffraction-based immunosensors have used silicon wafer chips that were in contact with analytecontaining solutions and required washing and drying before analysis by a simple reader (3 ). The dotLab system uses a plastic consumable, the dotLab Sensor, with an integrated prism situated below the flow channel so that the light source interrogates the diffraction grating without passing through the bulk solution. Previous studies suggest that this total internal reflection scheme allows for 95% of the laser intensity to be measured, whereas in a nontotal internal reflection set-up only 5% is measured (4 ). Moreover, with this configuration, our system monitors biomolecule binding in real time and in complex media.We tested the beta prototype of the dotLab instrument, which incorporates the core dot technology in an integrated package intended for in-house and con...

Development of a Qualitative Sequential Immunoassay for Characterizing the Intrinsic Properties of Circulating Cardiac Troponin I

“…The amplified dot Immunoassay could minimally detect 2 g/L (approximately 84 pmol/L) of cTnI (on the basis of detection of 55-94 aa by 3E3 mAb). N-terminal pro-B-type natriuretic peptide (NT-proBNP) has been minimally detected by dot Immunoassay at 31.25 ng/L (approximately 4 pmol/L) (23 ).…”

“…The assays were performed at room temperature in dotLab Sensors with avidin surface chemistry (Axela) on a dotLab System (Axela) (23 ). We developed different assays as listed below and repeated each assay at least twice.…”

“…The subsequent capture of the analyte (purified analyte or serum) alone or bound to a detector antibody increases the average height of the surface pattern and causes an increase in the diffraction intensity (DI) signal that is recorded in real time. The DI signal is directly related to the size and quantity of the bound complex (22,23 ).…”

“…All binding events were independent from each other, and each was evaluated separately. For data comparison among different experiments, we normalized DI signals of the compared binding step against the ⌬ of bt-8I-7 binding (as "normalized DI"), thus minimizing intersensor variation (such ratio comparison is typical and has been reported previously (23 )). …”

“…The protocol uses diffractive optics technology, bringing together the techniques of grating-based light diffraction and immobilized capture surfaces to allow sequential analysis of the captured antigen (22,23 ). This method can directly measure circulating cTnI to determine if it is phosphorylated or degraded at either the N-or C-terminus.…”