Optical plasmonic biosensor for leukemia detection
Biosensors are devices that can be used to specifically detect biomolecules of interest in applications such as clinical analysis, drug discovery, and food safety. In general, biosensors have several advantages over conventional diagnostic techniques. For instance, they are label-free and can be used to perform real-time detection. Furthermore, they consume only small quantities of expensive reagents and ingredients, and highly skilled personnel are not required for their operation. These properties therefore contribute to a considerable reduction of cost per test, while providing rapid detection results. Although biosensors have been successfully commercialized, at present very few devices are actually used in hospitals or the point-of-care. This is because of various issues associated with the devices, e.g., bulkiness, poor detection limits, non-specific binding (NSB), and high costs.
Work in the biosensor field is currently expanding along two main paths: miniaturization and improvement of the limit of detection (LOD) in complex fluids. Attempts to miniaturize optical biosensors, for example, have included a surface plasmon resonance (SPR) biosensor integrated with a cell phone for the detection of β2 microglobulin (a marker for cancer, inflammatory disorders, and kidney disease).1 The LOD of biosensors is often limited by NSB and can be improved by modifying the sensor surface chemistry or the detection assay approach.2
To address the aforementioned issues with biosensors, we have developed a novel optical biosensor that has great potential for miniaturization. In our new biosensor we use long-range surface plasmon polariton (LRSPP) waveguides. LRSPPs are surface plasmon waves that can propagate—upon excitation—over appreciable lengths along a metal slab or stripe (waveguide). As biomolecules accumulate on the surface, the attenuation of the waveguide changes, which thus alters the power output of the structure. This process forms the basis of biosensing, i.e., by tracking events in real time on the waveguide surface. It is possible to functionalize the metal surface with different specific recognition elements (e.g., antibodies) to selectively identify and capture a molecule of interest.4 We have also demonstrated the use of the LRSPP sensor for B-cell leukemia detection. To conduct this demonstration, we have introduced an innovative assay—based on Protein G—which significantly reduces NSB.5
In our work, we fabricated LRSPP biosensors (see Figure 1) for B-cell leukemia detection as a series of gold (Au) straight waveguides (SWGs) embedded in Cytop (a fluoropolymer with a low refractive index that matches biologically compatible fluids). The top Cytop cladding is partially etched into the Au level for fluidic access, and we excite the LRSPPs by butt-coupling the fiber to the input of the waveguide. A characteristic of B-cell leukemia is the production of a disproportionate distribution of kappa and lambda light chain immunoglobulins (IgGκ and IgGλ, respectively) in blood, and this fact is exploited in leukemia diagnosis. In normal patient serum, the IgGκ:IgGλ ratio ranges from 1.4 to 2.0. In leukemia patients, however, the ratio deviates significantly (either to increased IgGκ or IgGλ).
We tested the sera—with known (a priori) immunoglobulin content—from three patients. We categorized these as normal serum, high-kappa serum, and high-lambda serum as they had IgGκ:IgGλ ratios of about 1.7, 12.7, and 6.9, respectively. For waveguide functionalization, we chose Protein G because it is known to bind specifically to the fragment crystallizable (Fc) region of immunoglobulin and thus promotes an upward orientation of antibodies. A common biosensor strategy is to attach a recognition element onto the surface, followed by the injection of the analyte. We have, however, introduced an innovative ‘reverse’ approach. In this method, we first immobilize the patient serum onto the Protein G-functionalized surface and subsequently inject the recognition antibodies (see Figure 2). The Protein G surface also acts as an ‘immunological filter,’ as it primarily captures IgGs out of the pool of serum proteins, and thus reduces NSB in the following recognition step. Results from two example tests for IgGκ and IgGλ in the high-kappa serum are shown in Figure 3.
We have also compared our final experimental results with known data from densitometric measurements (see Figure 4). We performed three separate tests on each patient sample to get a good sense of the repeatability. Overall, we find that our sensor has the capability of differentiating between the normal, high-kappa, and high-lambda serum samples. Our actual IgGκ:IgGλ ratios, however, differ from the densitometric data. This is a matter of ongoing investigation.
In summary, we have developed and tested a novel optical LRSPP biosensor for B-cell leukemia detection. Our results indicate that the sensor is suitable for clinical diagnostic applications. Our LRSPP biosensor can also be miniaturized, and it is therefore a fast and low-cost solution to various biosensor detection problems. In addition, we have demonstrated an innovative approach to immunoglobulin identification in serum. This method reduces NSB, irrespective of the biosensor type. Our future work on the sensor will include modifications to the fluidic channel so that we can achieve better solution mixing, and ultimately, we will miniaturize the sensor.
The authors gratefully acknowledge National Sciences and Engineering Research Council of Canada (BiopSys Network) for funding. Ritch Dusome and other employees of Cisco (Ottawa) are also thanked for a philanthropic donation in support of this work. In addition, we gratefully acknowledge Ewa Lisicka-Skrzek, Sa'ad Hassan, and Anthony Olivieri for their assistance in conducting these experiments.
University of Ottawa
Oleksiy Krupin recently obtained his PhD from the University of Ottawa. His research interests involve the testing of new biosensor types. For his PhD, he dealt with modification of surface chemistry and the design of experimental protocols to investigate the capabilities of a novel optical plasmonic biosensor.
Mount Sinai Hospital
University of Toronto
University of Ottawa