David A. Bergstein

Label-free and dynamic detection of biomolecular interactions for high-throughput microarray applications

Direct monitoring of primary molecular-binding interactions without the need for secondary reactants would markedly simplify and expand applications of high-throughput label-free detection methods. A simple interferometric technique is presented that monitors the optical phase difference resulting from accumulated biomolecular mass. As an example, 50 spots for each of four proteins consisting of BSA, human serum albumin, rabbit IgG, and protein G were dynamically monitored as they captured corresponding antibodies. Dynamic measurements were made at 26 pg/mm2 SD per spot and with a detectable concentration of 19 ng/ml. The presented method is particularly relevant for protein microarray analysis because it is label-free, simple, sensitive, and easily scales to high-throughput.

Quantification of DNA and protein adsorption by optical phase shift

A primary advantage of label-free detection methods over fluorescent measurements is its quantitative detection capability, since an absolute measure of adsorbed material facilitates kinetic characterization of biomolecular interactions. Interferometric techniques relate the optical phase to biomolecular layer density on the surface, but the conversion factor has not previously been accurately determined. We present a calibration method for phase shift measurements and apply it to surface-bound bovine serum albumin, immunoglobulin G, and single-stranded DNA. Biomolecules with known concentrations dissolved in salt-free water were spotted with precise volumes on the array surface and upon evaporation of the water, left a readily calculated mass. Using our label-free technique, the calculated mass of the biolayer was compared with the measured thickness, and we observed a linear dependence over 4 orders of magnitude. We determined that the widely accepted conversion of 1 nm of thickness corresponds to approximately 1 ng/mm(2) surface density held reasonably well for these substances and through our experiments can now be further specified for different types of biomolecules. Through accurate calibration of the dependence of thickness on surface density, we have established a relation allowing precise determination of the absolute number of molecules for single-stranded DNA and two different proteins.

Label-free multiplexed virus detection using spectral reflectance imaging

We demonstrate detection of whole viruses and viral proteins with a new label-free platform based on spectral reflectance imaging. The Interferometric Reflectance Imaging Sensor (IRIS) has been shown to be capable of sensitive protein and DNA detection in a real time and high-throughput format. Vesicular stomatitis virus (VSV) was used as the target for detection as it is well-characterized for protein composition and can be modified to express viral coat proteins from other dangerous, highly pathogenic agents for surrogate detection while remaining a biosafety level 2 agent. We demonstrate specific detection of intact VSV virions achieved with surface-immobilized antibodies acting as capture probes which is confirmed using fluorescence imaging. The limit of detection is confirmed down to 3.5 × 10(5)plaque-forming units/mL (PFUs/mL). To increase specificity in a clinical scenario, both the external glycoprotein and internal viral proteins were simultaneously detected with the same antibody arrays with detergent-disrupted purified VSV and infected cell lysate solutions. Our results show sensitive and specific virus detection with a simple surface chemistry and minimal sample preparation on a quantitative label-free interferometric platform.

Resonant Cavity Imaging: A Means Toward High-Throughput Label-Free Protein Detection

The resonant cavity imaging biosensor (RCIB) is an optical technique for detecting molecular binding interactions label free at many locations in parallel that employs an optical resonant cavity for high sensitivity. Near-infrared light centered at 1512.5 nm couples resonantly through a Fabry-Perot cavity constructed from dielectric reflectors (Si/SiO2), one of which serves as the binding surface. As the wavelength is swept using a tunable laser, a near-infrared digital camera monitors cavity transmittance at each pixel. A wavelength shift in the local resonant response of the optical cavity indicates binding. Positioning the sensing surface with respect to the standing wave pattern of the electric field within the cavity controls the sensitivity with which the presence of bound molecules is detected. Transmitted intensity at thousands of pixel locations is recorded simultaneously in a 10 s, 5 nm scan. An initial proof-of-principle setup has been constructed. A test sample was fabricated with 25,100-mum wide square features, each with a different density of 1-mum square depressions etched 12 nm into the SiO2 surface. The average depth of each etched region was found with 0.05 nm rms precision. In a second test, avidin, bound selectively to biotin conjugated bovine serum albumin, was detected.

Self-referencing substrates for optical interferometric biosensors

Optical interference is a powerful technique for monitoring surface topography or refractive index changes in a thin film layer. Reflectance spectroscopy provides label-free biosensing capability by monitoring small variations in interference signature resulting from optical path length changes from surface-adsorbed biomolecules. Spectral reflectance data can be acquired either by broad wavelength illumination and spectroscopy at a single point, thus necessitating scanning, or by varying the wavelength of illumination and imaging the reflected intensity allowing for acquisition of a spectral image of a large field of view simultaneously. In imaging modalities, intensity fluctuations of the illuminating light source couple into the detected signal, increasing the noise in measured surface profiles. This article introduces a simple technique for eliminating the effects of illumination light power fluctuations by fabricating on-substrate self-reference regions to measure and normalize for the incident intensity, simplifying the overall platform for reflection or transmission-based imaging biosensors. Experimental results demonstrate that the sensitivity performance using self-referencing is equivalent or better than an optimized system with an external reference.

Hyperspectral Fourier transform spectrometer for reflection spectroscopy and spectral self-interference fluorescence microscopy

A hyperspectral Fourier transform spectrometer has been developed for studying biological material bound to optically reflecting surfaces. This instrument has two modes of operation: a white-light reflection mode and a spectral self-interference fluorescence mode. With the combined capability, information about the conformation of an ensemble of biomolecules may be determined. To the best of our knowledge, ours is the first report of this hybrid white-light reflection, spectral self-interference fluorescence measurement with any type of hyperspectral imager. The measurement technique is presented along with a full description of the system, including theoretical performance projections. Proof-of-principle measurements of artificial samples are shown, and the results are discussed.

Synergetic chemiluminescence and label-free dual detection for developing a hepatitis protein array.

A dual detection system for protein arrays is presented that combines label-free detection by optical interference with chemiluminescence. A planar protein array that targets hepatitis B surface antigen is developed. Surface densities for individual antibody spots are quantitated using optical interference prior to use. Target binding (10 ng/ml) is detected label-free. Target binding (1 ng/ml) is detected by both optical interference and chemiluminescence with the inclusion of secondary antibodies. Binding results using both methods are found to be directly proportion to the capture probe density measured initially. The dual detection system provides the analytical utility of optical interference detection with the established clinical utility of chemiluminescence detection.

Label-free and high-throughput screening of biomolecular interactions

We present a simple label-free multi-analyte detection technique that is easily scalable for high-throughput screening. We have shown a sensitivity of 20 pg/mm2 and a minimum detectable antibody concentration of 15 ng/ml for a specific antigen.

Silicon substrtates with buried distributed bragg reflectors for biosensing

High quality silicon substrates with buried Bragg reflectors previously developed within our group for improved photodetectors, have enabled a new biosensing modality. The goal of this technique is to detect the presence of biomolecules binding to a surface coated with different localized capturing agents. Such ability yields information about the affinity of the biomolecules under test for the molecules on the capturing surface. Capturing surfaces such as microarrays featuring thousands of different binding locations with different fixed capturing agents offer the greatest amount of information and hence benefit. Information about the affinity between molecules of interest such as particular proteins or DNA strands, yields great benefit to a number of applications in biological research and may soon become applicable in medical diagnostics as well. Microarrays are a technology that accomplishes just this [1]. Thousands or hundreds of thousands of different capturing agents are fixed in localized spots or features on the microarray surface, typically glass. Molecules under test are first affixed with a fluorescent label and then introduced to the microarray surface. A fluorescent scanner then scans the surface with a laser and records the fluorescent signal with a PMT to determine the amount of binding at each location. But microarray technology has its limitations despite its wide spread success. Namely, the need to fluorescently label the molecules of interest can preclude microarrays from many applications where affixing a label to the test molecules may not be practical or possible. In addition, proteins in particular may suffer from altered binding properties once the label is affixed. For these reasons, a label-free sensing technology, such as described here, would be preferable. Optical detection of low level molecular binding is achieved using an interferometric technique that benefits from a resonant cavity enhancement. The resonant cavity is formed between two facing planar Bragg reflectors bearing the following qualities: high reflectivity, low loss, exceptional smoothness, and cost effectiveness. Light enters the cavity through the back of the first Bragg reflector. The wavelength is swept in time using a wavelength tunable laser source. When the wavelength satisfies the resonant condition of the cavity for a particular location, the cavity builds up local energy that couples through and is recorded by a camera pixel corresponding to that location, or alternatively a photodiode in a photodiode array. The high finesse of the cavity afforded by the high reflectivity of the reflectors causes the resonance to be highly sensitive to slight changes within the cavity. Biological agents binding to the capturing agents at different locations within the cavity modulate the local resonant condition within the cavity. As the wavelength is swept in time and images recorded on the camera, resonant wavelength-transmission curves are recorded for each cavity locations. Shifts in these curves indicate binding. This measurement can be made simultaneously at thousands of locations, in minutes.

Optical phase to biological mass conversion for label-free interferometric sensing methods

The conversion between the detected optical phase and the biological mass accumulated on the surface is determined using a label-free interferometric technique. The results demonstrate a linear phase/mass relationship for DNA and protein microarrays.