Proceedings Volume 3913

In-Vitro Diagnostic Instrumentation

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Proceedings Volume 3913

In-Vitro Diagnostic Instrumentation

View the digital version of this volume at SPIE Digital Libarary.

Volume Details

Date Published: 11 April 2000
Contents: 6 Sessions, 23 Papers, 0 Presentations
Conference: BiOS 2000 The International Symposium on Biomedical Optics 2000
Volume Number: 3913

Table of Contents

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Table of Contents

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  • Advances in Noninvasive Diagnostics
  • Cellular Diagnostics I
  • Cellular Diagnostics II
  • Advanced Ultrasensitive Diagnostic Systems
  • Advanced Biosensor-Based Instrumentation I
  • Advanced Biosensor-Based Instrumentation II
  • Advanced Ultrasensitive Diagnostic Systems
Advances in Noninvasive Diagnostics
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Continuing evolution of in-vitro diagnostic instrumentation
The synthesis of analytical instrumentation and analytical biochemistry technologies in modern in vitro diagnostic instrumentation continues to generate new systems with improved performance and expanded capability. Detection modalities have expanded to include multichip modes of fluorescence, scattering, luminescence and reflectance so as to accommodate increasingly sophisticated immunochemical and nucleic acid based reagent systems. The time line graph of system development now extends from the earliest automated clinical spectrophotometers through molecule recognition assays and biosensors to the new breakthroughs of biochip and DNA diagnostics. This brief review traces some of the major innovations in the evolution of system technologies and previews the conference program.
Progress in noninvasive optical diagnostics
It may look odd that a session on noninvasive diagnostks is scheduled as a part of an in— vitro diagnostic instrumentation symposium. Hopefully, NI diagnostics will grow to the extent that we will have several sessions on different aspects of it in future meetings. In this talk I will discuss the general field of non-invasive diagnostics from industry perspective with examples on what are the advances in hemoglobin and glucose measurements. The other two talks in this session will deal with NI optical devices for bilirubin (SpectRx) and Hb/Hct (In-Line Diagnostics) that just achieved regulatory clearance and are ready for the market. A tissue oxygenation photon density waves photometer for tissue oxygenation measurements (ISS Instruments) and a brain oxygen saturation instrument (Hamamatsu) are available commercially for research purposes. It was quite encouraging to see how a novel technology as optical coherence tomography made its way from the research lab to a clinical product for ophthalmic applications, the workshop arranged on this topic was quiet interesting. SPIE had several sessions on the past few years on optical biopsy, laser interaction with tissue, optical coherence tomography, photoacoustics interactions in tissues and photon migrations in tissues. Some of these techniques will eventually find their way to the hospital floor, the ICU and the doctors office. Some of the constant participants in this annual BIOS/SPIE meeting as Chance, Jacques, Gratton, Alfano, Tromberg, Delpy, Fujimuto, Ezzat, and others have laid the scientific foundation of optical diagnostics, practical applications may be soon on the horizon.
Bilirubin measurements in neonates
Gregory J. Newman
Infant Jaundice is a physiologic condition of elevated bilirubin in the tissue that affects nearly 60 percent of all term newborns and virtually 100 percent of premature infants. The high production of bilirubin in the newborn circulatory system and the inability of the immature liver to process and eliminate it case the condition. When the bilirubin levels rise, it starts to deposit in the baby's skin and in the brain. The deposits in the brain can cause neurologic impairment and death. The BiliCheck is a handheld, battery-powered device that measures the level of jaundice non-invasively using BioPhotonics at the point of care. The result is displayed on an LCD screen immediately, so physicians can now make treatment decision without waiting for results to return from the lab. The BiliCheck System has been marketed worldwide since April of 1998 and has received FDA clearance for use in the USA on pre-photo therapy infants in March of 1999.
Cellular Diagnostics I
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Application of advanced cytometric and molecular technologies to minimal residual disease monitoring
Minimal residual disease monitoring presents a number of theoretical and practical challenges. Recently it has been possible to meet some of these challenges by combining a number of new advanced biotechnologies. To monitor the number of residual tumor cells requires complex cocktails of molecular probes that collectively provide sensitivities of detection on the order of one residual tumor cell per million total cells. Ultra-high-speed, multi parameter flow cytometry is capable of analyzing cells at rates in excess of 100,000 cells/sec. Residual tumor selection marker cocktails can be optimized by use of receiver operating characteristic analysis. New data minimizing techniques when combined with multi variate statistical or neural network classifications of tumor cells can more accurately predict residual tumor cell frequencies. The combination of these techniques can, under at least some circumstances, detect frequencies of tumor cells as low as one cell in a million with an accuracy of over 98 percent correct classification. Detection of mutations in tumor suppressor genes requires insolation of these rare tumor cells and single-cell DNA sequencing. Rare residual tumor cells can be isolated at single cell level by high-resolution single-cell cell sorting. Molecular characterization of tumor suppressor gene mutations can be accomplished using a combination of single- cell polymerase chain reaction amplification of specific gene sequences followed by TA cloning techniques and DNA sequencing. Mutations as small as a single base pair in a tumor suppressor gene of a single sorted tumor cell have been detected using these methods. Using new amplification procedures and DNA micro arrays it should be possible to extend the capabilities shown in this paper to screening of multiple DNA mutations in tumor suppressor and other genes on small numbers of sorted metastatic tumor cells.
Multispectral bacterial identification
Michael A. Tanner, William J. Coleman, Christine L. Everett, et al.
A multi spectral optical technique was developed to simultaneously classify individual bacterial cells within mixed populations. Multi spectral Bacterial Identification (mBID) combines innovations in microscopy with a software analysis program to measure and characterize the fluorescence signals from multiplexed 16S ribosomal RNA probes hybridized to populations of different bacteria. Software was developed to identify individual bacteria at the level of species within these mixed populations. TO test the feasibility of mBID, we examined the fluorescence emissions from a mixture of probes specific for individual species of known bacteria from the American Type Culture Collection. Currently, up to seven species can be detected simultaneously by fluorescence microscopy. An eighth signal was reserved for a universal probe to control for fluorescence intensity. mBID can also be used to identify uncultured microorganisms. We plan to couple this new multi spectral technology to existing identification technologies that utilize 16S rRNA sequence alignment. Using this integrated identification protocol, bacteria that may be associated with chronic conditions will be identified first by analyzing their 16S rDNA sequences and then by visualizing them with flourescent probes hybridized to their 16S rRNA in situ.
Cyclic peptidase substrates for fluorescent analysis of Caspase 3 enzyme activity
Anthony P. Guzikowski, Christina Shipp, Rachel A. Howard, et al.
Cyclic Caspase 3 peptidase substrates derived from the fluorophore Rhodamine 110, as well as their cleavage products, were prepared by step-wise synthesis, using three independent methods. 3D energy minimized structural analysis as well as Mass Spectral analysis indicate that the cyclic substrates can bind positively charged cations. Enzymatic assays indicate that the substrates are cleaved by the Caspase 3 enzyme. Cellular assays showed cell specific staining of apoptosis induced cell lines. Application of these substrates to the high-throughput screening analysis of apoptosis induction in whole cells was developed.
Magnetic field effects on human lymphocytes: methodological assessments and experimental evidences
Marziale Milani, Monica Ballerini, Giuliana Baroni, et al.
The results are discussed of a systematic investigation on the electromagnetic field exposure consequences on human lymphocytes. These artificial fields have intensities comparable with the Earth magnetic field one, and are used for exposures up to 4 days. Different and complementary techniques are used to safely assess the consequences of ElectroMagnetic Fields (EMF) on the cells; in particular morphology, metabolism and population dynamics are investigated. The recourse to ultra microscopy, pressure monitoring in sealed bottles, atomic mass spectroscopy. Far IR Fourier Transform and cytofluorimetry techniques give a good insight in the EMF induced changes. A statistically significant deviation of irradiated samples with respect to the control ones are reported. A critical analysis and a survey of similar experiments reported in literature lead us to the exam of the experimental set up with attention to the geometry of the irradiation system. Finally the role of different magnetic field detectors in the reproducibility of the experiments will be carefully discussed.
Cellular Diagnostics II
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Nucleic acid in-situ hybridization detection of infectious agents
Curtis T. Thompson
Limitations of traditional culture methods and newer polymerase chain reaction (PCR)-based methods for detection and speciation of infectious agents demonstrate the need for more rapid and better diagnostics. Nucleic acid hybridization is a detection technology that has gained wide acceptance in cancer and prenatal cytogenetics. Using a modification of the nucleic acid hybridization technique known as fluorescence in-situ hybridization, infectious agents can be detected in a variety of specimens with high sensitivity and specificity. The specimens derive from all types of human and animal sources including body fluids, tissue aspirates and biopsy material. Nucleic acid hybridization can be performed in less than one hour. The result can be interpreted either using traditional fluorescence microscopy or automated platforms such as micro arrays. This paper demonstrates proof of concept for nucleic acid hybridization detection of different infectious agents. Interpretation within a cytologic and histologic context is possible with fluorescence microscopic analysis, thereby providing confirmatory evidence of hybridization. With careful probe selection, nucleic acid hybridization promises to be a highly sensitive and specific practical diagnostic alternative to culture, traditional staining methods, immunohistochemistry and complicated nucleic acid amplification tests.
Bench-top self-contained laser-scanning cytometer with liquid handling capabilities for arbitrary fluid-based clinical diagnostic assays
Edward M. Goldberg, David M. Heffelfinger, Ning Sizto, et al.
Out of necessity, small bench top clinical diagnostic instruments have been limited in on-board features that can be packed into the dimensions of a stand-alone instrument. This often reduces the functionality of these machines to a narrow range of tests and can also substantially increase the complexity and cost of the consumable components. We have addressed these limitations in a novel bench top clinical device. With confocal optics in combination with an autofocusing method we are able to target and image a thin layer of cells for analysis of shape and spectral properties. Due to the non-CCD based detection method, the system has an optimized depth of focus that allows for detection of cells while rejecting bulk background fluorescence, thus greatly reducing background signal and increasing signal-to-noise. The flexibility in the cartridge design allows for a wide variety of assays, including multi- step reagent mixing and incubation, and multiple assays on a single sample. Further, use of volumetric capillaries allows the determination of absolute cell counts in specified volumes, eliminating the need for counting references. The multi-PMT detection takes advantage of assays using multiple stains. Bar code reading allows for sample identification and other information. On-board communications interfacing allows flexible LIS options, remote software upgrading, and detailed development and debug information access. We present laser-scanning cytometer with a small footprint that includes on-board liquid handling and facilitates a diverse set of clinical assays, while improving user-safety and ease of use.
Fully automated three-dimensional microscopy system
Russell L. Kerschmann M.D.
Tissue-scale structures such as vessel networks are imaged at micron resolution with the Virtual Tissue System (VT System). VT System imaging of cubic millimeters of tissue and other material extends the capabilities of conventional volumetric techniques such as confocal microscopy, and allows for the first time the integrated 2D and 3D analysis of important tissue structural relationships. The VT System eliminates the need for glass slide-mounted tissue sections and instead captures images directly from the surface of a block containing a sample. Tissues are en bloc stained with fluorochrome compounds, embedded in an optically conditioned polymer that suppresses image signals form dep within the block , and serially sectioned for imaging. Thousands of fully registered 2D images are automatically captured digitally to completely convert tissue samples into blocks of high-resolution information. The resulting multi gigabyte data sets constitute the raw material for precision visualization and analysis. Cellular function may be seen in a larger anatomical context. VT System technology makes tissue metrics, accurate cell enumeration and cell cycle analyses possible while preserving full histologic setting.
Advanced Ultrasensitive Diagnostic Systems
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Laser scattering method applied to determine the concentration of alfa 1-antitrypsin
Bibiana Doris Riquelme, Patricia Foresto, Juana R. Valverde, et al.
An optical method has been developed to find (alpha) 1- antitrypsin unknown concentrations in human serum samples. This method applies light scattering properties exhibited by initially formed enzyme-inhibitor complexes and uses the curves of aggregation kinetics. It is independent of molecular hydrodynamics. Theoretical approaches showed that scattering properties of transient complexes obey the Rayleigh-Debie conditions. Experiments were performed on the Trypsin/(alpha) 1-antitrypsin system. Measurements were performed in newborn, adult and pregnant sera containing (alpha) 1-antitrypsin in the Trypsin excess region. The solution was excite by a He-Ne laser beam. SO, the particles formed during the reaction are scattering centers for the interacting light. The intensity of the scattered light at 90 degrees from incident beam depends on the nature of those scattering centers. Th rate of increase in scattered intensity depends on the variation in size and shape of the scatterers, being independent of its original size. Peak values of the first derivative linearly correlate with the concentration of (alpha) 1-antitrypsin originally present in the sample. Results are displayed 5 minutes after the initiation of the experimental process. Such speed is of great importance in the immuno-biochemistry determinations.
Optically active nanoparticles for ultrasensitive detection and spectroscopy
Dustin J. Maxwell, John T. Krug II, Shuming Nie
Recent research in our lab has identified a new class of metal nanoparticles that are highly efficient for surface- enhanced optical spectroscopy. By coupling nanoparticles with surface-enhanced Raman scattering (SERS), surface optical processes can be enhanced by 14 to 15 orders of magnitude. This enormous enhancement allows the optical detection of single molecules at room temperature. However, in a standard silver colloid only 1 out of 100 nanoparticles is SERS-active. In this report, a new methodology based on size-selective filtration is demonstrated. This size- selective fractionation procedure yields an enriched colloid that can be used to prepare highly efficient nanoparticle thin films.
Issues in enzyme-based metal ion biosensing in complex media
Richard B. Thompson, Hui-Hui Zeng, Michele Loetz, et al.
In developing fluorescence-based biosensors for rapidly determining metal ions such as zinc or copper in complex media such as sea water, serum, cerebrospinal fluid, or the interior of a cell, several issues must be considered. Among these are the selectivity, sensitivity, ease of calibration, speed of response, reversibility, stability, and the ease of immobilization onto a solid substrate. While the first three have been dealt with in the design of our transducer molecule, apocarbonic anhydrase II, the others remain to be considered. In this paper we examine the stability of the apoprotein to storage at various temperatures and pHs with a view to establishing storage conditions and lifetimes in operating environments for sensor transducers. Similarly, we immobilized a storage conditions and lifetimes in operating environments for sensor transducers. Similarly, we immobilized a fluorescent-labeled apo-CA variant on quartz to determine its sped, sensitivity, and kinetics of response.
Putting the pieces together: contribution of fluorescence polarization assays to small-molecule lead optimization
Susan M. Keating, Jim Marsters, Maureen Beresini, et al.
Fluorescence polarization assays with both purified receptor and intact cells have been developed to assess potency and selectivity of antagonists of the interaction of the lymphocyte receptor, LFA-1, and its endothelial ligand, ICAM-1. Fluorescein isothiocyanate conjugated small molecule probes were optimized for use in binding assay with LFA-1 and a closely related receptor, MAC-1. In the assays, the antagonists compete with the fluorescent probe for binding to the receptor. This enables the determination of IC50 and consequently Ki values of the antagonists for each of the receptors. Routine use of polarization assay with tranfected cells, in addition to purified receptors, has become feasible with the availability of sensitive plate readers that are able to detect 1 nM fluorescent probe in 15 (mu) l sample volumes with good signal to noise. These measurements aid in the iterative synthesis of more potent and selective compounds.
In-vitro measurements of light transmission parallel and perpendicular to the collagen orientation in tendons
Binod KC, Kevin R. Forrester, Dave Irvine-Halliday, et al.
Collimated light transmission studies of bovine tendon have been carried out to show the collagen waveguide effect. A monochromator/lamp apparatus was used to irradiate tissue samples and the transmitted light intensity was measured parallel and perpendicular to the collagen fiber orientation. The intensity parallel to the collagen fibers was at least twice that seen with perpendicular propagation. This indicates there is less scattering parallel to the fibers and light waveguiding is present for photon scattering. Also, absorption of light due to hemoglobin around 550 nm and due to water at 980nm was more prominent for parallel than perpendicular propagation. The light may travel either through the collagen fibril or fiber bundles or through the interstitial matrix. Sequential tests during tissue dehydration were performed and it was found that the transmitted light intensity increased with dehydration. This suggest that light may not be traveling through the interstitial matrix where water is the major component. Water may be acting as a reflection boundary for the light that is passing through the fibril or fiber bundles. Collagen waveguiding may be utilized to elucidate the collagen structure. Also, tissue water content could be measured from the transmission profiles. These may be of use in diagnosis and repair of connective tissues.
Advanced Biosensor-Based Instrumentation I
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Next fundamental steps for optical DNA biosensors
Lisa Henke, James H. Watterson, Ulrich J. Krull
Methods for the detection of pathogenic organisms and genetic mutations have evolved to use the selective recognition chemistry offered by nucleic acid hybridization. The immobilization of single-stranded DNA as a 'receptor' onto various solid surfaces that are suitable for optical sensor development is a fundamental issue that determines aspects of selectivity, sensitivity and speed. This manuscript presents an evaluation of various immobilization strategies to attach ssDNA to surfaces, the control of such immobilization to design optical and thermodynamic properties of monolayer films, and suggests approaches for optimization of analytical figures of merit and the potential for quantitative analysis.
Planar waveguide biosensors for nucleic acid hybridization reactions
James N. Herron, Stacy zumBrunnen, Jian-xun Wang, et al.
Oligonucleotide probes derived from (1) the T3 RNA polymerase promoter sequence (T3) and (2) prostate-specific antigen messenger RNA (PSA) were prepared and labeled with a red-emitting fluorescent dye (Cy5). The complimentary oligonucleotides were prepared and labeled with biotin. Initially, a feasibility study was performed in which the hybridization rate of the T3/anti T3 Oligonucleotide pair was examined. Specifically, biotinylated anti T3 was immobilized to a neutravidin-coated waveguide and solutions containing increasing concentrations of Cy5-labeled T3 were injected into the biosensor. Fluorescence emission was detected with an evanescent wave imaging fluorometer. The hybridization reaction proceeded rapidly with a significant amount of binding occurring during the first 5 minutes. A Michaelis-Menton kinetics model was used to analyze hybridization rate data and gave values of 78 nanomolar for the apparent affinity of the hybridization reaction and 1.4 picomolar for the analytical sensitivity of the hybridization assay. In subsequent studies the hybridization rate of the PSA/anti PSA oligonucleotide pair was examined. Biotinylated anti PSA was immobilized to the waveguide and solutions containing increasing concentration of Cy5-labeled PSA were injected into the biosensor. The hybridization rate observed for formation of the PSA/anti PSA pari was comparable to the high rates observed for the T3/anti T3 pari. Lastly, the selectivity of the biosensor was examined using an Oligonucleotide probe derived from human glandular kallikrein, which exhibits a high degree of homology to PSA. The two Oligonucleotide probes only differed in 7 out of 20 positions. Interestingly, the hybridization rate observed for Cy5-labeled hGK was very low - not statistically different from the non-specific binding rate of the hybridization assay.
Advanced Biosensor-Based Instrumentation II
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Development of an automated handheld immunoaffinity fluorometric biosensor
Terry E. Phillips, C. Brent Bargeron, Richard C. Benson, et al.
A new immunoaffinity fluorometric biosensor has been developed for detecting and quantifying aflatoxins, a family of potent fungi-produced carcinogens that are commonly found in a variety of agriculture products. They have also been cited as a biological agent under weapons development. The handheld, self-contained biosensor is fully automatic, highly sensitive, quick, quantitative, and requires no special storage. Concentration from 0.1 parts per billion to 50 ppb can be determined in less than 2 minutes with a 1 ml sample volume. Higher concentrations can be determined by simply reducing the sample volume. The device operates on the principles of immunoaffinity for specificity and fluorescence for a quantitative assay. The analytic procedure is flexible so that other chemical and biological analytes could be detected with minor modifications to the current device.
Multiphoton up-converting phosphors for use in rapid immunoassays
R. Sam Niedbala, Timothy L. Vail, Hans Feindt, et al.
There exists increasing demand for rapid in-vitro diagnostic test capable of detecting pathogens, disease markers, and drugs. Many currently marketed technologies are limiting due to their analytical sensitivity or the number of analytes detected in a single assay. Presented is the use of up- converting phosphors as reporters in rapid point-of-care diagnostic assays using a lateral flow format in a nitrocellulose matrix. Up-converting Phosphor Technology (UPT) utilizes lanthanide-doped ceramic particles that possess anti-Stokes shift emission. These material absorb multiple IR photons and emit dopant-dependent spectra in the visible range. Their signature spectra allow the development of sensitive, multi-analyte diagnostic test. Because no biological matrix up-converts in the IR, the sensitivity of the assay is enhanced, being determined by the affinity of the biological constituents and the level of non-specific binding of the particle reporter. Feasibility data presented below represent a wide range of molecular weight analytes, including drugs of abuse, proteins such as cardiac markers, and pathogenic microorganisms.
Immunoaffinity-based phosphorescent sensor platform for the detection of bacterial spores
Peter F. Scholl, C. Brent Bargeron, Terry E. Phillips, et al.
Consideration of emergency response plans to an attack with biological weapons such as anthrax spores has spawned renewed interest in the development of inexpensive, rapid, and sensitive field portable sensors for use by non- specialists. The conceptual feasibility of such a device is demonstrated via the immunoaffinity capture of spores of the anthrax simulant B. globigii on a column followed by their washing, elution and phosphorescent detection. Spores are generically detected via the rapid extraction of dipicolinic acid (DPA) followed by its chelation with terbium to yield a phosphorescent complex. Chemical, thermal and mechanical methods of DPA extraction were evaluated. Simple extraction in HNO3 released up to 5 percent of the spore weight as DPA within 60 seconds. Extraction in H2O liberated 7 percent of the spore weight as DPA. Sonication with glass beads in H2O for 45 seconds released up to 4 percent of the spore weight as DPA. It is estimated that implementation of these techniques will permit development of a device requiring 3-5 minutes per analysis with a limit of detection on the order of 500 ng spore/mL. This approach is not intended to replace more specific methods of analysis. However, it is proposed for consideration as an inexpensive, simple and rapid means of spore detection by non-specialists in emergency situations.
Evanescent planar waveguide detection of biological warfare simulants
David M. Sipe, Kenneth P. Schoonmaker, James N. Herron, et al.
An evanescent planar waveguide Mark 1.5 instrument was used to detect simulants of biological warfare agents; ovalbumin (OV), MS2 bacteriophage, BG, and Erwinia herbicola (EH). Polyclonal tracer antibodies were labeled with the fluorescent dye, Cy5. Discrete bands of polyclonal capture antibodies were immobilized to a polystyrene planar waveguide with molded integral lenses. An ST-6 CCD camera was used for detection. OV. MS2 and BG were detected in a simultaneous 3 by 3 array; with a total of nine measurements within 6 minutes. EH was analyzed in a separate array. Results were evaluate dat the US Army Joint Field Trials V, at the Dugway Proving Grounds. Over a 10 day period, 32 unknown samples were analyzed daily for each simulant. Detection limits: OV 10 ng/ml, MS2 107 pfu/ml, BG 105 cfu/ml. EH was detectable at 5 X 105 cfu/ml. Overall false positives were 3.0 percent. Therefore, the Mark 1.5 instrument, with a parallel array of detectors, evanescent flourescent excitation, and CCD imaging provides for rapid, sensitive, and specific detection of biological warfare agent simulants.
Noninvasive fluorescence-based instrumentation for cancer and precancer detection and screening
In this paper, we review our research in the use of UV and visible native fluorescence emission and excitation spectroscopy for the detection of cancer and precancer. We discuss some of the spectroscopic signatures indicative of the presence of cancer and precancer. We describe three generations of instruments being developed to extent optical biopsy technology into the clinical environment as both a screening tool and as a diagnostic aide suitable for gynecological, gastro-intestinal tract, oral cavity, brain and breast.
Advanced Ultrasensitive Diagnostic Systems
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Fluorescent measurements in whole blood and plasma using red-emitting dyes
Omoefe O. Abugo, Petr Herman, Joseph R. Lakowicz
We have determined the fluorescence characteristics of albumin blue 670 and Rhodamine 800 in plasma and blood in order to test the feasibility of making direct fluorescence sensing measurements in blood. These dyes were used because of their absorption in the red/NIR where absorption by hemoglobin is minimized. Front face illumination and detection was used to minimize absorption and scattering during measurement. Fluorescence emission was observed for these dyes in plasma and blood. Attenuation of the fluorescence emission was observed in blood because of hemoglobin absorption. Using frequency domain fluorometry, we recovered the expected lifetime parameters for both dyes in blood and plasma. We were able to quantify HSA concentrations using changes in the mean lifetime of AB670, a dye previously shown to bind preferentially to HSA. Rh800 concentrations in plasma and blood were also determined using modulation sensing. Anisotropy measurements revealed high Anisotropy for these dyes in plasma and blood. It also showed an increase in the anisotropy of AB670 with increase in HSA concentration in the presence of red blood cells. These results indicate that qualitative and quantitative fluorescence measurements can be made directly in blood without the need to process the blood.