This PDF file contains the front matter associated with SPIE Proceedings Volume 9310, including the Title Page, Copyright information, Table of Contents, Invited Panel Discussion, and Conference Committee listing.

Optical waveguide biosensors based on silicon-on-insulator (SOI) have been extensively investigated owing to its various advantages and many potential applications. In this article, we demonstrate a novel highly sensitive biosensor based on cascaded Mach-Zehnder interferometer (MZI) and ring resonator with the Vernier effect using wavelength interrogation. The experimental results show that the sensitivity reached 1,960 nm/RIU and 19,100 nm/RIU for sensors based on MZI alone and cascaded MZI-ring with Vernier effect, respectively. A biosensing application was also demonstrated by monitoring the interaction between goat and antigoat immunoglobulin G (IgG) pairs. This integrated high sensitivity biosensor has great potential for medical diagnostic applications.

Shiga toxin-producing Escherichia coli (STEC) poses a serious threat to human health through the consumption of contaminated food products, particularly beef and produce. Early detection in the food chain, and discrimination from other non-pathogenic Escherichia coli (E. coli), is critical to preventing human outbreaks, and meeting current agricultural screening standards. These pathogens often present in low concentrations in contaminated samples, making discriminatory detection difficult without the use of costly, time-consuming methods (e.g. culture). Using multiple signal transduction schemes (including novel optical methods designed for amphiphiles), specific recognition antibodies, and a waveguide-based optical biosensor developed at Los Alamos National Laboratory, we have developed ultrasensitive detection methods for lipopolysaccharides (LPS), and protein biomarkers (Shiga toxin) of STEC in complex samples (e.g. beef lysates). Waveguides functionalized with phospholipid bilayers were used to pull down amphiphilic LPS, using methods (membrane insertion) developed by our team. The assay format exploits the amphiphilic biochemistry of lipoglycans, and allows for rapid, sensitive detection with a single fluorescent reporter. We have used a combination of biophysical methods (atomic force and fluorescence microscopy) to characterize the interaction of amphiphiles with lipid bilayers, to efficiently design these assays. Sandwich immunoassays were used for detection of protein toxins. Biomarkers were spiked into homogenated ground beef samples to determine performance and limit of detection. Future work will focus on the development of discriminatory antibodies for STEC serotypes, and using quantum dots as the fluorescence reporter to enable multiplex screening of biomarkers.

Human Immunodeficiency Virus (HIV) has been the subject of intense research for more than three decades as it causes an uncurable disease: Acquired Immunodeficiency Syndrome, AIDS. In the pursuit of a medical treatment, RNAtargeted small molecules are emerging as promising targets. In order to understand the binding kinetics of small molecules and HIV RNA, association (k_a) and dissociation (k_d) kinetic constants must be obtained, ideally for a large number of sequences to assess selectivity. We have developed Aqueous Array Imaged Reflectometry (Aq-AIR) to address this challenge. Using a simple light interference phenomenon, Aq-AIR provides real-time high-throughput multiplex capabilities to detect binding of targets to surface-immobilized probes in a label-free microarray format. The second generation of Aq-AIR consisting of high-sensitivity CCD camera and 12-μL flow cell was fabricated. The system performance was assessed by real-time detection of MBNL1-(CUG)₁₀ and neomycin B - HIV RNA bindings. The results establish this second-generation Aq-AIR to be able to examine small molecules binding to RNA sequences specific to HIV.

Arrayed Imaging Reflectometry (AIR) is a highly sensitive label-free biosensor which can be used to detect hundreds of antigens on a single substrate. The signal monitored with AIR is the light intensity of an angled beam reflected off of a flat substrate which is composed of a protein-reactive film on a thermally grown silicon oxide layer. If the angle, wavelength, and polarization of the incident light beam is fixed, a near-zero reflectance condition can be obtained by adjusting the thickness of the thermally grown oxide. In a typical AIR biosensing experiment, antibodies are printed (using a piezoelectric microarrayer) on top of the oxide layer to create a minimum reflectance condition. If the substrate is exposed to a complex solution (such as serum), the patterned antibodies bind to their specific targets increasing the effective spot thickness, which perturbs the anti-reflective condition and causes a measurable signal increase. One of the main considerations with AIR is evaluating and controlling the bioactivity and efficiency of antibody immobilization after printing, since these factors significantly affect the dynamic range and limit of detection. Here, we present preliminary experiments towards using microgel nanoparticles as a simple and customizable construct to deposit antibodies on biosensor surfaces. This method can be generalized to work with other microarray technology formats, including those that are not label-free.

Optofluidic schemes of inhibition, transport and activation by carrier molecules through cell membrane have interesting applications. Through plasmonic excitation of nanoparticles integrated in microfluidic channel, we observe cell membrane structural changes. Related phenomena are studied in situ in a microfluidic channel via fluorescence imaging. Detailed analysis is carried out to understand the possible application of this scheme in optically induced transport and expression of cell membrane protein. Optical properties of the cells undergoing plasmonic transport are monitored and correlated to cell expression assay. Plasmonic charge transport and optical transmission are measured in the microfluidic lab-on-chip along with in-situ imaging.

We demonstrate the first use of smartphone spectrophotometry for readout of fluorescence-based biological assays. We evaluated the smartphone fluorimeter in the context of a fluorescent molecular beacon (MB) assay for detection of a specific nucleic acid sequences in a liquid test sample. The capability of distinguishing a one-point mismatch is also demonstrated by detecting single-base mutation in target nucleic acids. Our approach offers a route towards portable biomolecular assays for viral/bacterial pathogens, disease biomarkers, and toxins.

In this study, bio-sensing pads are proposed to capture living cells, which are fabricated on cover glasses by cross-linking proteins/antibodies using laser induced photochemistry. The biological functions of the cross-linked protein/antibody were verified by capturing Staphylococcus aureus (S. aureus), Leptospira, and red blood cells (RBCs), separately, with associated protein/antibody sensing pads. The experimental results show that S. aureus were bound on GFP-AcmA’ pad after minutes of incubation and phosphate buffered saline (PBS) rinsing. No binding was observed with reference pad made of neutral bovine serum albumin (BSA). Second, A-type RBCs were chosen as the model cell to demonstrate the blood typing feasibility of the anti-A pad in microchannel. The A-type RBCs were captured only by the anti-A pad, but not the reference pad made of BSA. The same experimental model was carried out on the Leptospira, which stuck on the blood serum pad after PBS rinsing, but not BSA pad. This study provides a potential platform for simple and direct detection of living full cells without culture that could be used in point-of-care settings.

The study utilized thermophoresis, the directed motion of molecules in a temperature gradient to quantify DNA and proteins for point-of-care applications. Because the direction and speed of thermophoretic motion is dependent on the size, charge, and conformation of the molecules, the binding between molecules can induce changes in their thermophoretic motion. To quantify biomolecules using thermophoresis, we mixed fluorescently-labeled capture probes with samples and then used an infrared laser to create a temperature gradient in the solution. By adding a small fraction of polymers to the buffer solution, we accumulated the fluorescent probes in a temperature gradient using the thermophoretic effects. The thermophoretic motion of the fluorescent probes significantly changed as the target molecules bind to the specially designed capture probes. Consequently, the level of the thermophoretic accumulation, which was determined by the spatial distribution of fluorescent probes, could be used to quantify molecules. This method functioned well even when the buffer contained 10% serum, which suggested that the detection was resistant to the interferences from the molecules in serum. The thermophoresis-based detection method developed in this study only requires a laser and an epi-fluorescence microscope during the detection. Unlike many other commonly seen biosensing methods, quantifying molecules using thermophoresis does not need any fluid channels or pumps for washing away unbound molecules during the detection process. In addition, the detection does not rely on any micro- or nanofabricated chips. In short, this thermophoresis-based biosensing method can be a simple, robust, and sensitive method for quantifying proteins and DNA.