We are developing fluorescence-free interferometric biosensors for the early detection of epithelial ovarian cancer
(EOC) and prognosis of acute lymphocytic leukemia (ALL). We can detect potential early markers for EOC (CA125,
human epididymus protein 4, osteopontin) spiked into serum as well as elevated CA125 in EOC patient serum. For ALL
prognosis we are focusing on three intracellular protein markers (p73, p57/Kip2, and p15/Ink4b), the down-regulation of
any two being indicative of a more aggressive cancer. We have detected p15 and p57 spiked into buffer and are
preparing to test positive and negative control lysates from bone marrow biopsies.
We present a new type of protein microarray called the Land-contrast (LC) BioCD in which imaging contrast is
induced by a patterned substrate rather than by patterned protein. This is realized by etching spot patterns in a
silicon wafer. On the spot region the silicon dioxide thickness is 140 nm and on the land it is 77 nm. The
spot and the land have equal reflectance but opposite interferometric quadrature responses for protein layer.
Protein is evenly immobilized on the entire chip and detected by reflectometry. Therefore there is no need for
protein printing, nor spectrometers, nor high angles nor polarization control to image the surface-bound protein.
The LC BioCD can facilitate research on protein microarrays.
In solid-support immunoassays, the transport of target analyte in sample solution to capture molecules on the sensor
surface controls the detected binding signal. Depletion of the target analyte in the sample solution adjacent to the
sensor surface leads to deviations from ideal association, and causes inhomogeneity of surface binding as analyte
concentration varies spatially across the sensor surface. In the field of label-free optical biosensing, studies of
mass-transport-limited reaction kinetics have focused on the average response on the sensor surface, but have not
addressed binding inhomogeneities caused by mass-transport limitations. In this paper, we employ Molecular
Interferometric Imaging (MI2) to study mass-transport-induced inhomogeneity of analyte binding within a single protein
spot. Rabbit IgG binding to immobilized protein A/G was imaged at various concentrations and under different flow
rates. In the mass-transport-limited regime, enhanced binding at the edges of the protein spots was caused by depletion
of analyte towards the center of the protein spots. The magnitude of the inhomogeneous response was a function of
analyte reaction rate and sample flow rate.
Molecular Interferometric Imaging (MI2) is a sensitive detection platform for direct optical detection of immobilized
biomolecules. It is based on inline common-path interferometry combined with far-field optical imaging. The substrate is a simple thermal oxide on a silicon surface with a thickness at or near the quadrature condition that produces a π/2 phase shift between the normal-incident wave reflected from the top oxide surface and the bottom silicon surface. The presence of immobilized or bound biomolecules on the surface produces a relative phase shift that is converted to a far-field intensity shift and is imaged by a reflective microscope onto a CCD camera. Shearing interferometry is used to remove the spatial 1/f noise from the illumination to achieve shot-noise-limited detection of surface dipole density profiles. The lateral resolution of this technique is diffraction limited at 0.4 micron, and the best longitudinal resolution is 10 picometers. The minimum detectable mass at the metrology limit is 2 attogram, which is 8 antibody molecules of size 150 kDa. The corresponding scaling mass sensitivity is 5 fg/mm compared with 1 pg/mm for typical SPR sensitivity. We have applied MI2 to immunoassay applications, and real-time binding kinetics has been measured for antibody-antigen reactions. The simplicity of the substrate and optical read-out make MI2 a promising analytical assay tool for high-throughput screening and diagnostics.
Specific protein concentrations in human body fluid can serve as diagnostic markers for some diseases, and a quantitative and high-throughput technique for multiplexed protein detection would speed up diagnosis and facilitate medical research. For this purpose, our group developed the BioCD, a spinning-disc interferometric biosensor on which antibody is immobilized. The detection system adopts a common-path scheme making it ultra stable. The scaling mass sensitivity is below 10 pg/mm for protein surface density. A 25000-spot antibody BioCD was fabricated to measure the concentration of prostate specific antigen (PSA), a protein indicating prostate cancer if its level is high. Statistical analysis of our immunoassay results projects that the detection limit of PSA would reach 20 pg/ml in a 2 mg/ml background solution. For future prospects, a multiplexed BioCD can be produced for simultaneous diagnosis of diverse diseases. For instance, 100 markers above 200 pg/ml could be measured on a single disc given that the detection limit is inversely proportional to square root of the number of spots.
The biological compact disc (BioCD) is a sensitive detection platform that detects immobilized biomolecules on the
surface of a spinning disc by quadrature laser interferometry. Spinning-disc interferometry (SDI) has the advantage of
operating faraway from the 1/f system noise which has a 40 dB per octave slope, thus reducing the detection noise floor
by more than 50 dB compared to static interferometric detection techniques. Three quadrature classes of BioCD have
been previously reported: micro-diffraction, adaptive optical and phase contrast. In this paper, we introduce a new class
of BioCD, the in-line quadrature class, which has achieved a new level of simplicity and sensitivity. A silicon wafer
coated by a layer of SiO2 is used as a substrate for immobilized biomolecules. The thickness of the SiO2 layer is chosen
so that light reflected from the SiO2 surface on top and the silicon surface below is approximately in phase quadrature.
Protein molecules scatter the incident light, adding a phase shift linearly proportional to the mass density of the
immobilized protein, which is converted to a far-field intensity shift by quadrature interference. Patterning of protein is
achieved by spot printing with a jet printer, which produces protein spots 0.1 mm in diameter. We demonstrate the
sensitivity of the in-line quadrature BioCD by an equilibrium dose response experiment on a disc printed with 25,000
proteins spots with a detection limit of 1 ng/mL when divided into 32 virtual wells and treated as 32 separate assays.
This current performance is not a fundamental limit, and improvements in disc uniformity will enable scaling up to large
numbers of individual assays per disc.
We have developed a four-channel detection method on a protein-patterned BioCD that simultaneously measures
fluorescence, Rayleigh scattering and/or diffraction, and two interferometric channels in orthogonal quadratures: one that
measures differential phase and the other that measures direct phase. The latter two channels constitute label-free
interferometric protein detection, while fluorescence and Mie scattering detection provide complementary tools. The
BioCD is constructed as dielectric coated disks. Protein molecules patterned on dielectric films change the Fresnel
reflection coefficient of the films. The change is exhibited in two ways: the reflection coefficient and the phase are both
modulated. These are detected simultaneously by a split detector and designated as "amplitude" signal and "phase
contrast" (PC) signal. We are able to scan and image patterned proteins across an entire coated disk with high speed in
four channels. A single-analyte immunoassay shows strong correlation between the fluorescence channel and the
interferometry channel with a detection limit of 10 ng/ml in a complex protein background (rat lysate) concentration of 7
mg/ml.
The BioCD is a class of self-referencing interferometric optical biosensor that measures phase modulation from proteins
on a spinning disk. The optical detection of the patterns at high speed yields low noise floors far from 1/f noise. Two
scans of a disk before and after a 20 hour buffer wash are differenced yielding an rms surface height measurement error
of 45 pm corresponding to 5 femtograms of protein within a focal spot diameter. Simple area scaling relations are
derived that predict the performance of immunoassays as a function of well area. The scaling mass sensitivity of the
BioCD is determined to be 0.25 pg/mm under the conditions of an assay, with a metrology limit of the technique
between 0.05 to 0.1 pg/mm. The BioCD sensitivity is equivalent to the best reported surface mass sensitivity of surface
plasmon resonance sensors, and is achieved without resonant structures and hence is easy to fabricate and operate.
We previously introduced the biological compact disk (BioCD) as a sensitive detection platform to detect patterned biomolecules immobilized on the surface of a spinning disk. Spinning-disk interferometry allows high speed detection (10 microseconds per spot) of optical path length changes down to sub-nanometer scales
with high repeatability. The key to performing stable interferometry on a mechanically spinning disk is self-referencing: locking the phase of the signal and reference beams to quadrature (μ/2 phase difference) independent of mechanical vibrations or relative motion. Two quadrature classes of BioCD have been reported previously: the micro-diffraction class (MD-Class) and the adaptive optical class
(AO-Class) {Peng, 2004 #565; Varma, 2004 #440}. In this paper, we introduce a third class of BioCD, the Phase-Contrast-Class (PC-Class) BioCD. Protein is immobilized using photolithography on a disk in a 1024 spoke pattern. The edge of the printed protein pattern diffracts a focused laser beam that is detected in the Fourier plane with a split detector. The signal from the split detector is differenced, which plays a role in the electronic domain similar to that of a phase plate in optical phase contrast imaging. The PC-Class BioCD is simple in both theory and implementation, requiring no microstructure fabrication and no complex detection. Its potential in high
speed label-free biosensing is demonstrated by a two-analyte immunoassay that shows good rejection of nonspecific binding and low antibody cross-reactivity. Immunoassays were performed against IgG immunoglobulins with detection of bound analyte on pictogram level. To show the potential of scaling up to hundreds or thousands of analytes per disk, an experiment was also performed with small drops of protein solution.
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