This PDF file contains the front matter associated with SPIE Proceedings Volume 7167, including the Title Page, Copyright information, Table of Contents, Introduction (if any), and the Conference Committee listing.

Pathogen detection and analysis is critical for medicine, food safety, agriculture, public health and biosecurity. Many current microbial detection approaches are based on century-old culturing methods which, while reliable, are slow, provide relatively little information about the pathogens and are not adaptable to high throughput operations. Optical biodetection represents a potential alternative. Most ELISA and chromatography systems are based on optical methods that are also used for analysis of molecular interactions, such as DNA hybridization and protein-protein interactions (e.g. microarrays or SPR biosensors). Various optical biosensor platforms have been developed that have many of the characteristics essential for modern pathogen molecular analysis including sensitivity, speed of analysis, multi-channel capability, relative simplicity and low cost. Here we provide several examples of the use of optical biosensor technology for pathogen detection and analysis including high throughput DNA microarray analysis, SPR-based rapid direct detection of bacterial toxins, CCD-based fluorescent activity analysis of microbial toxins and a simple ECL-based CCD detection system. However, while effective for molecular analysis, most of these technologies are not as sensitive as traditional culturing methods for detecting microorganisms. There is a need to combine optical biosensors with traditional methods to speed culture-based detection and to provide more information regarding the pathogens.

Cost-effective disease diagnosis in resource-limited settings remains a critical global health challenge. Qualitative rapid tests based on lateral flow technology provide valuable screening information, but require relatively expensive confirmatory tests and generally lack quantitation. We report on a fluorescence technology that combines low cost instrumented readout with passive pumping in a disposable cartridge. The detection system utilizes a novel waveguide illumination approach in conjunction with commercial CMOS imagers. Total instrument cost in production are projected to be around $100 This cost structure and instrument ease of use will enable use in point-of-care settings, outside of centralized laboratories. The system has been used for detection and analysis of proteins, antibodies, nucleic acids, and cells. Here we will report first on our development of a multiplexed, array-based serology assay for HIV and common AIDS co-infections. Data will be presented for HIV/HCV antibody testing in human serum samples. In addition, we will present data on the use of the system for sensitive detection of bacterial RNA. Current detection limit for the model multiplexed RNA sandwich assay is 1 femtomolar target RNA. Finally, a high magnification version of the system is used to image immunostained human T cells.

The BioHawk is a highly automated aerosol collector and 8-channel wet assay bio-identifier integrated into a single man-portable case. Samples may be transferred automatically from the aerosol collector to the bio-identifier portion under software recipe control, or from a sample port provided for grab sample boluses. Eight parallel wet assays are performed within a disposable plastic coupon, and each coupon may be reused 10 or more times. All reagents and fluids needed for the baseline sandwich immunoassay protocols are contained within the coupon in lyophilized form: On first use, reagents are automatically hydrated. The system is designed for long-term unattended operation and user intervention requirements are minimal to none.

In the last decade surface plasmon resonance (SPR) biosensors have made great strides both in terms of technology and its applications. SPR biosensors have become a central tool for study of molecular interactions and have been widely used for detection of chemical and biological analytes. Food analysis belongs to major areas of potential applications of SPR biosensors. Therefore, numerous SPR biosensors for detection of analytes implicated in food safety (e.g. pathogens, toxins, drug residues, vitamins, hormones, chemical contaminants, and allergens) have been developed. This paper reviews recent developments in the field of SPR biosensors for food safety, in particular, for detection of foodborne pathogens and toxins.

It is important to screen our food supply for pathogen contaminations. Current methods to screen for bacterial contamination involve using costly reagents such as antibodies or PCR reagents or time-costly growth in cultures. There is need for portable, real-time, multiplex pathogen detection technology that can predict the safety of food where it is produced or distributed. Surface plasmon resonance (SPR) imaging is a sensitive, label-free method that can detect the binding of an analyte to a surface due to changes in refractive index that occur upon binding. It can be used for label-free detection of the presence of potential pathogens. Simultaneous fluorescence molecular imaging on the other side of the biochip can be used to ascertain pathogen status or functional state which may affect its potential danger to humans or animals. We are designing and testing hybrid microfluidic biochips to detect multiple pathogens using a combination of SPRI and fluorescence imaging. The device consists of an array of gold spots, each functionalized with a peptide targeting a specific pathogen. This peptide biosensor array is enclosed by a PDMS microfluidic flow chamber that delivers a magnetically concentrated sample to be tested. An SPR image is taken from the bottom of the biochip. Image analysis is used to quantify the amount of pathogen (both live and dead) bound to each spot. Since PDMS is very transmissive to visible light, an epi-fluorescence image is taken from the top of the biochip. Fluorescence imaging determines the live:dead ratio of each pathogen using an inexpensive SYTO 9(R)-Propidium Iodide assay. The volume of sample that the biochip can analyze is small, so possible pathogens are pre-concentrated using immunomagnetic separation. Functionalized magnetic particles are bound to pathogens present in the sample, and a magnet is used to separate them from the bulk fluid.

We have developed a waveguide-based optical biosensor for the sensitive and specific detection of biomarkers associated with disease. Our technology combines the superior optical properties of single-mode planar waveguides, the robust nature of functionalized self-assembled monolayer sensing films and the specificity of fluorescence sandwich immunoassays to detect biomarkers in complex biological samples such as serum, urine and sputum. We have previously reported the adaptation of our technology to the detection of biomarkers associated with breast cancer and anthrax. However, these approaches primarily used phospholipid bilayers as the functional film and organic dyes (ex: AlexaFluors) as the fluorescence reporter. Organic dyes are easily photodegraded and are not amenable to multiplexing because of their narrow Stokes' shift. Here we have developed strategies for conjugation of the detector antibodies with quantum dots for use in a multiplex detection platform. We have previously evaluated dihydroxylipoic acid quantum dots for the detection of a breast cancer biomarker. In this manuscript, we investigate the detection of the Bacillus anthracis protective antigen using antibodies conjugated with polymer-coated quantum dots. Kinetics of binding on the waveguide-based biosensor is reported. We compare the sensitivity of quantum dot labeled antibodies to those labeled with AlexaFluor and demonstrate the photostability of the former in our assay platform. In addition, we compare sulfydryl labeling of the antibody in the hinge region to that of nonspecific amine labeling. This is but the first step in developing a multiplex assay for such biomarkers on our waveguide platform.

A porous silicon waveguide with integrated grating coupler is demonstrated as a new platform for portable detection of chemical and biological molecules. The two-layer porous silicon waveguide is formed by electrochemical etching and a photoresist grating is fabricated directly on the waveguide core by means of electron beam lithography. Angle-resolved reflectance measurements reveal distinct peaks corresponding to the guided mode. A 0.420° reflectance shift was observed upon 16-base DNA hybridization, which was more than a factor of 5 larger than the observed reflectance shift after exposure to a mismatched DNA sequence.

Photonic crystal (PhC) microcavities present multiple advantages for rapid, accurate, label-free, and sensitive detection. But their principle of operation (observation of a peak in transmission) makes their integration in serial arrays difficult. Here we report on multiple resonant cavities coupled to a single photonic crystal waveguide. The device configuration consists of a PhC waveguide with a defect line along which light is guided. Several resonant microcavities, created by modifying the radius of a hole adjacent to the defect line, are coupled to the waveguide. This PhC device, operating as a multi-channel sensor, maintains the advantages of the PhC microcavities and allows for serial arrays: Light is globally transmitted through the waveguide, except for the wavelengths corresponding to the resonant modes of the microcavities. The transmission spectrum shows as many dips as there are cavities. Simulations show that the sensitivity of such structures allows the detection of single particles -typically a virus. Preliminary results show the fabrication and characterization of a double-channel structure with small defects as a solvent sensor.

This work focuses on the development of a proof-of-concept optical sensor design that incorporates an amine-functionalized polyacrylamide hydrogel into a 1D porous silicon (PSi) photonic crystal. The PSi acts as both a template and a transducer capable of detecting morphological and dielectric changes in the incorporated hydrogel structure. Free radical copolymerization of acrylamide (AAm) and N-(3- aminopropyl)-methacrylamide (NA) monomers was utilized to form copolymer chains with a controlled concentration of nucleophilic amine moieties. These amine groups facilitated chemical cross-linking of the copolymer chain to generate hydrogel networks. A molar fraction of >2 mol% of NA monomer was needed to facilitate a visibly gelatinous hydrogel in a 5 wt% polymer solution. Addition of sodium formate (chain transfer agent) during copolymer synthesis facilitated decreased copolymer chain length and improved infiltration of the copolymer into the p-type PSi mesoporous sensor (pore diameters ~20-30 nm). Controlled cross-linking of the copolymer chains was completed with using glutaraldehyde, as a model system, to form a hydrogel network that could be optically monitored by the incorporated PSi sensor. These results lay foundation for extending this versatile methodology towards the design of an affinity based complimentary target-probe system to create a hybrid target-responsive hydrogel-PSi chemical sensor.

Highly sensitive optical techniques, capable of detecting very small quantities of specific proteins in a label-free format, offer great promise for pathogen detection because they avoid the complexity, expense, and process time associated with the use of secondary reporter elements. Arrayed Imaging Reflectometry (AIR) is one of the simplest label-free methodologies, combining laser reflectance imaging of a thermally oxidized silicon chip with standard microarray printing technology to create a platform with the potential to identify and quantify 100's of target proteins in a matter of minutes. This technique exploits a reflectance zero condition that is formed when s-polarized light strikes the surface of a silicon wafer with a single-layer oxide coating. In the vicinity of this deep reflectance minimum, picometer-scale variations in film thickness (surface relief) can be imaged directly in a reflected laser signal imaged with a CCD camera. By directly arraying probe molecules onto this substrate, minute changes in the optical thickness of each spot, corresponding to binding of the target of interest, can be measured. Array size is limited only by the resolution of the imaging system and the array printer, enabling complex protein signatures, indicative of specific pathogens or disease states to be measured in a biosample. The cost-effectiveness of a low-complexity substrate and reader, combined with the short assay times associated with label-free detection make AIR a promising new technology for pathogen and toxic exposure assessment.

Arrayed Imaging Reflectometry, or "AIR", is a new label-free technique for detecting proteins that relies on bindinginduced changes in the response of an antireflective coating on the surface of a silicon ship. Because the technique provides high sensitivity, excellent dynamic range, and readily integrates with standard silicon wafer processing technology, it is an exceptionally attractive platform on which to build systems for detecting proteins in complex solutions. In our early research, we used AIR chips bearing secreted receptor proteins from enteropathogenic E. coli to develop sensors for this pathogen. Recently, we have been exploring an alternative strategy: Rather than detecting the pathogen directly, can one immobilize antigens from a pathogen, and employ AIR to detect antibody responses to those antigens? Such a strategy would provide enhanced sensitivity for pathogen detection (as the immune system essentially amplifies the "signal" caused by the presence of an organism to which it responds), and would also potentially prove useful in the process of vaccine development. We describe herein preliminary results in the application of such a strategy to the detection of antibodies to human papillomavirus (HPV).

Rapid and accurate detection of biomolecules is important for medical diagnosis, pharmaceuticals, homeland security, food quality control, and environmental protection. A simple, low cost and highly sensitive label-free optical biosensor based on opto-fluidic ring resonator (OFRR) has been developed that naturally integrates microfluidics with ring resonators. The OFRR employs a piece of fused silica capillary with a diameter around 100 micrometers. The circular cross section of the capillary forms the ring resonator and light repeatedly travels along the resonator circumference in the form of whispering gallery modes (WGMs) through total internal reflection. When the capillary wall is as thin as a couple of micrometers (< 4 μm), an evanescent field of the WGMs exists at the OFRR inner surface and interacts with the sample when it flows through the OFRR. In order to detect the target molecules with high specificity, the OFRR inner surface is functionalized with receptors, such as antibodies, peptide-displayed bacteriophage or oligonucleotide DNA probes. The WGM spectral position shifts when biomolecules bind to the OFRR inner surface and change the local refractive index, which provides quantitative and kinetic information about the biomolecule interaction near the OFRR inner surface. The OFRR has been successfully demonstrated for detection of various types of biomoelcuels. Here, we will first introduce the basic operation principle of the OFRR as a sensor and then application examples of the OFRR in the detection of proteins, disease biomarkers, virus, DNA molecules, and cells with high sensitivities will be presented.

The development of rapid assays for detection of microbial pathogens in complex matrices is needed to protect public health due to continued outbreaks of disease from contaminated foods and water. An Escherichia coli O157:H7 detection assay was designed using a robotic, fluorometric assay system. The system integrates optics, fluidics, robotics and software for the detection of foodborne pathogens or toxins in as many as four samples simultaneously. It utilizes disposable fiber optic waveguides coated with biotinylated antibodies for capture of target analytes from complex sample matrices. Computer-controlled rotation of sample cups allows complete contact between the sample and the waveguide. Detection occurs via binding of a fluorophore-labeled antibody to the captured target, which leads to an increase in the fluorescence signal. Assays are completed within twenty-five minutes. Sample matrices included buffer, retentate (material recovered from the filter of the Automated Concentration System (ACS) following hollow fiber ultrafiltration), spinach wash and ground beef. The matrices were spiked with E. coli O157:H7 (10³-10⁵ cells/ml) and the limits of detection were determined. The effect of sample rotation on assay sensitivity was also examined. Rotation parameters for each sample matrix included 10 ml with rotation, 5 ml with rotation and 0.1 ml without rotation. Detection occurred at 104 cells/ml in buffer and spinach wash and at 10⁵ cells/ml in retentate and ground beef. Detection was greater for rotated samples in each matrix except ground beef. Enhanced detection of E. coli from large, rotated volumes of complex matrices was confirmed.

Lab on a chip has found many applications in biological and chemical analysis, including pathogen detections. Because these labs on chips involve handling of fluids at the microscale, surface tension profoundly affects the behavior and performance of these systems. Through careful engineering, controlled liquid-liquid or liquid-gas interfaces at the microscale can be formed and used in many interesting applications. In this talk, I will present our work on applying such interfaces to microsensing. These interfaces are created at hydrophobic-hydrophilic boundaries formed within microfluidic channels and pinned by surface tension. We have designed and fabricated a few microsensing techniques including chemical and biological sensing using dissolvable micromembranes in microchannels, chemical and biological sensing at liquid crystals interfacing either air or aqueous solutions, and collection of gaseous samples and aerosols through air-liquid microfludic interfaces. I will next introduce on-chip microlenses and microlens arrays for optical detection, including smart and adaptive liquid microlenses actuated by stimuli-responsive hydrogels, and liquid microlenses in situ formed within microfluidic channels via pneumatic control of droplets.

We report an in situ formed tunable liquid microlense array and its applications for enhancing dynamic lab-on-a-chip performance. The de-ionized water microlenses are intrinsically formed via liquid-air interfaces of liquid droplets at T-shaped junctions of octadecyltrichlorosilane(OTS) treated polymerized isobornyl acrylate(poly(IBA)) microchannels., and can be separately tuned in focal lengths by pneumatic manipulation. Via the tunable microlenses, excitation light is dynamically focused onto the fluorescent fluidic sample, thus the fluorescence emission signal for detection is amplified. We have further shown the potential for surface reaction study at microfluidic interfaces by the microlense array.

A multi-analyte diagnostic system based on a novel microflow cytometer is under development as a portable, fielddeployable sensor for environmental monitoring and for rapid point-of-care and on-site diagnosis of exposure to biothreat agents. The technology relies on a unique method for ensheathing a sample stream in continuous flow past an illuminated interrogation region. This sheathing approach efficiently focuses particles in the interrogation region of the fluidic channel and minimizes clogging by complex samples. Fluorescently coded microspheres provide the capability for highly multiplexed assays. In this report, separation of six microsphere sets was demonstrated with determination of immunoassays on three of the six sets; comparison to the commercial platform was made.

Food-borne and waterborne microbial pathogens are a potential problem in biowarfare and public health. Such pathogens can affect the health, combat readiness, and effectiveness of the warfighter in a battlefield environment and present potential threats to the civilian population through intentional or natural contamination of food and water. Conventional procedures to detect and identify microbial pathogens in food, water, and other materials can take days to perform and may provide inconclusive information. Research at the University of South Florida's Advanced Biosensors Laboratory (ABL) focuses on development of sample processing procedures and biosensor-based assays for rapid detection of biothreat agents. Rapid processing methods, including use of an automated concentrator of microorganisms in water, have been developed for complex matrix samples including ground beef, apple juice, produce, potable water and recreational water, enabling such samples to be directly tested by biosensor assays for target analytes. Bacillus atrophaeus spores and other bacteria can be concentrated from potable and recreational water at low levels with a dead-end hollow-fiber ultrafiltration concentration system. Target bacteria recovered by these processing procedures can be identified by evanescent wave, fiber optic biosensors or other detection platforms. Fiber optic biosensor assays have been improved to include subsequent PCR analysis and viability determination of captured target bacteria using broth enrichment and/or ATP luminescence.

Our team has developed polyethylene glycol (PEG)-modified, self-assembled monolayers (SAMs) for biological detection on either planar or spherical substrates, which resist non-specific binding while facilitating specific ligand attachment. The preparation and characterization of these thin films, their validation against B. anthracis protective antigen (PA) in a sandwich assay format, and the application of these thin films for quantitative analysis of several medically interesting targets (breast cancer, tuberculosis, and influenza) will be shown.

We report a method for improving the sensitivity of label-free optical biosensors based on in-situ synthesis of DNA probes within porous silicon structures. The stepwise attachment of up to 15mer probes inside 30 nm mesopores was accomplished through a series of phosphoramidite reactions. In this work, a porous silicon waveguide was utilized as the sensor structure. Synthesis of DNA probe, as well as sensing of target DNA, was verified by monitoring the change in effective refractive index of the porous silicon waveguide through angle-resolved attenuated total reflectance measurements. The average resonance shift per oligo of 0.091° during stepwise synthesis corresponds to surface coverage slightly less than 50%, according to theoretical models. When compared with the traditional method of direct attachment of pre-synthesized oligonucleotide probes, the sequential phosphoramidite method resulted in an approximately four-fold increase in DNA probe attachment. This increased surface coverage by DNA probes increases the likelihood of target molecule binding, leading to improved sensitivity for bio-molecule detection. Exposure to a 50&mgr;M solution of target 8-base DNA in deionized water produced a 0.4236° change in the waveguide resonance angle. Nanomolar detection limits for small molecule sensing are realizable with this sensor scheme.

Staphylococcus aureus is a leading cause of human illness, and has developed the remarkable ability to resist the bactericidal capabilities of many of the world's leading antibiotics (i.e. MRSA). In an effort to enable rapid detection and treatment of MRSA infections, we have developed a DNA detection technology termed the NanoLantern(TM). The NanoLantern(TM) biosensor technology is based on the simple immobilization of a fluorophore-terminated DNA hairpin onto a gold chip. This produces a label-free sensor that allows for a positive response to be obtained without extensive processing of the sample, saving cost and increasing accuracy. We will also discuss a newly developed method of partial gene analysis, used to develop a DNA hairpin probe that is capable of detecting the presence of the mecR gene, a gene necessary for methicillin resistance to be present in S. aureus, with 100% sequence specificity. The successful incorporation of this probe into the NanoLantern(TM) platform, along with the concomitant development of the paired PCR assay has allowed for the successful detection of methicillin-resistance directly from a culture of S. aureus. These results represent an important step forward in terms of developing the ability to rapidly and effectively detect the presence of antibiotic resistance in bacterial infections.

We have been developing an array-based DNA biosensor, in which DNA oligonucleotides are readily immobilized onto Au surfaces via terminal thiol groups to serve as probes for the detection of oligonucleotides in solution. One common strategy to minimize steric effects and non-specific oligonucleotide-surface interactions derived from a high surface probe density employs thiol molecules as spacers. In this study, we tested nine different alkanethiol molecules with the goal of identifying a relationship between the chemical structure of each molecule and its performance as a spacer molecule. Among the nine different spacers, 4-mercapto-1-butanol and 1-decanethiol were found to yield the optimal performance as spacers, while spacers with a bulky tert-butyl group or a ring structure (2-methyl-2-propanethiol, cyclohexyl mercaptan and thiophenol) yielded poorer performance. Spacers with a short, branched carbon chain (1- mercapto-2-propanol and 3-mercapto-1,2-propanediol) provided an intermediate performance. These results suggest that DNA sensor response is highly sensitive to the chemical structure of the alkanethiol molecules that are employed as spacer molecules.

Enteric pathogens are a significant contaminant in surface waters used for recreation, fish and shellfish harvesting, crop irrigation, and human consumption. The need for water monitoring becomes more pronounced when industrial, agricultural, and residential lands are found in close proximity. Fecal contamination is particularly problematic and identification of the pollution source essential to remediation efforts. Standard monitoring for fecal contamination relies on indicator organisms, but the technique is too broad to identify the source of contamination. Instead, real-time PCR of mitochondrial DNA (mtDNA) is an emerging method for identification of the contamination source. Presented herein, we evaluate an alternative technology, the compact Bead Array Sensor System (cBASS®) and its assay approach Fluidic Force Discrimination (FFD), for the detection of mtDNA. Previously, we achieved multiplexed, attomolar detection of toxins and femtomolar detection of nucleic acids in minutes with FFD assays. More importantly, FFD assays are compatible with a variety of complex matrices and therefore potentially applicable for samples where the matrix would interfere with PCR amplification. We have designed a triplex assay for the NADH gene found in human, swine, and bovine mtDNA and demonstrated the specific detection of human mtDNA spiked into a waste water sample.

We present a biosensing platform that uses spatial electroluminescent (EL) illumination combined with charge-coupled device (CCD)-based detection for fluorescence measurements. The resulting EL-CCD detector platform was used to monitor different protease activities with substrates labeled for fluorescence resonance energy transfer (FRET)-based assays. The first uses a commercial FITC/DABCYL-SNAP-25 peptide substrate to monitor the activity of the light chain derivative (LcA) of botulinum neurotoxin A, achieving a limit of detection (LOD) of 1.25 nM (62 ng/ml). The second protease activity assay measured trypsin proteolysis using peptide substrates immobilized onto semiconductor quantum dot (QD) nanoparticles with a LOD of 6.2 nM trypsin (140 ng/ml). The specific ovomucoid inhibition of trypsin activity was also monitored. The highlighted studies clearly demonstrate the utility of the EL-CCD detector platform for monitoring fluorescent-based protease activity assays with potential healthcare applications, including point-of-care diagnostics.

One of suspect environmental endocrine disruptors that affect mouse male reproduction by altering the morphology of Sertoli cells and spermatogenic cells is phthalate. The effects of mono(2-ethylhexyl)phthalate (MEHP), one of metabolites of di(2-ethylhexyl)phthalate , on immature mouse testes in vivo were examined. We have recently shown that MEHP induced Sertoli cells necrosis and spermatogenic cells apoptosis in mice by TUNEL method, F-actin staining, and ultrastructural study, but there is no data for biochemical changing of testes due to those methods could not explore. To verify in detail of it, we conducted Raman spectroscopy study with 785 nm wavelength laser line, 50mW of laser power and 3 minutes of exposure time to analysis the MEHP-treated testicular tissue, which has been fixatived by 4% paraformaldehyde (PFA). Five weeks old (5 w.o) male mice were used in this experiment. As the results, the alterations were observed by Raman spectroscopy that there are significantly differences of DNA, actin filament, type IV collagen and amide I between control group (0 μM MEHP) and treatment group (100 μM MEHP). These results significantly support histology staining observation (such as the apoptotic spermatogenic cells which is associated with DNA fragmentation and F-actin disruption) and ultrastructural observation (such as mitochondria rupture and disintegration of nucleus membrane). Raman spectroscopy can be used for 4% PFA-fixatived tissue observation. However, we recommend that Raman spectroscopy may be able to be expanded as an armamentarium not just for the clarification of histology staining and ultrastructural study, but furthermore, it may be as a non-invasion assessment for screening animal tissue toxicity of chemical in future.

The construction of a specific, label-free, bacteria biosensor using porous silicon 1-D photonic crystals will be described. Bacteria resident on the surface of porous silicon act as scattering centers for light resonant with the photonic crystal; the diffusely scattered light possesses the optical spectrum of the underlying photonic crystal. Using a spectrometer fitted to a light microscope, the bacteria are imaged without using exogenous dyes or labels and are quantified by measuring the intensity of scattered light. In order to selectively bind and identify bacteria using porous Si, we use surface modifications to reduce nonspecific binding to the surface and to engineer bacteria specificity onto the surface. Bovine serum albumin (BSA) was adsorbed to the porous Si surface to reduce nonspecific binding of bacteria. The coatings were then chemically activated to immobilize polyclonal antibodies specific to Escherichia coli. Two E. coli strains were used in our study, E. coli DH5α and non-pathogenic enterohemorrhagic Escherichia coli (EHEC) strain. The nonpathogenic Vibrio cholerae O1 strain was used to test for antibody specificity. Successful attachment of antibodies was measured using fluorescence microscopy and the scattering method was used to test for bacteria binding specificity.