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We recently reported the development of a filament-antibody recognition assay (FARA), in which the presence of virions in solution initiates the formation of enzyme-linked immunosorbent assay (ELISA)-like antibody complexes. The unique features of this assay are that processing is achieved by motion of a filament and that, in the presence of a virus, antibody-virus complexes are coupled to the filament at known locations. In this work, we combine the unique features of this assay with a 638-nm laser-based optical detector to enable adaptive control of virus detection. Integration of on-line fluorescence detection yields approximately a five-fold increase in signal-to-noise ratio (SNR) compared to the fluorescence detection method reported previously. A one-minute incubation with an M13K07 test virus is required to detect 10(10) virionsml, and 40 min was required to detect 10(8) virionsml. In tests of the components of an adaptive strategy, a 30-min virus (3.3 x 10(10) virionsml) incubation time, followed by repositioning the filament-captured virus either within the detecting antibody chamber, (20 microg ml) or within the virus chamber, found an increase in signal roughly proportional to the cumulative residence times in these chambers. Furthermore, cumulative fluorescence signals observed for a filament-captured virus after repeated positioning of the filament within the virus chamber are similar to those observed for a single long incubation time. The unique features of the FARA-like design combined with online optical detection to direct subsequent bioprocessing steps provides new flexibility for developing adaptive molecular recognition assays.
DNA microarrays provide a method for determining the expression levels of thousands of genes simultaneously. However, the phenotypic complexity of brain tissue and cross-dilution of transcripts from different sources make it difficult to detect many of the low abundance RNA species. Furthermore, these experiments require significant amounts of starting material, which must often be amplified by one or two rounds of T7 amplification. We have developed a novel microarray probe with increased sensitivity. In this approach, PCR-generated microarray probes are end-ligated into redundant polymers and printed on standard arraying surfaces. These DNA polymer probes result in greatly improved sensitivity over classical monomer probes. Furthermore, polymer microarray sensitivity can be even further improved by incorporation of a biotin adapter into the first strand cDNA during reverse transcription and attachment of a gold particle (Genicon RLS, Invitrogen, CA) in a secondary reaction. This approach allowed us to reliably assess: expression of genes from < 5 microg of total RNA starting material without sample amplification. Finally, the resonance light scattering-labeled microarrays can be archived without fading, allowing re-scanning at a later time.
We present an analytic solution for the B1 field produced in a gapped toroidal cavity resonator designed as a probe for high field MRI. This resonator supports standing TEM waves, so its electric and magnetic fields are identical to those produced by a stationary planar current source with the same (constant) cross-section multiplied by a complex exponential propagation factor. An explicit expression for the field may therefore be found by solving Laplace's equation for the static potential, which is accomplished with a two-dimensional logarithmic conformal transformation algorithm. The equipotential curves are also the contours of the field strength B, and the B (vector) field at any point is directed along the contour passing through that point. With this information, we construct the solution by computing the angle made by the equipotential curve with the horizontal axis at each point, using this angle to analyze the B field into its x and y components, and adding the contributions from the current sources to obtain the magnitude and direction of B at each point in the region of interest. Some proposed extensions of this algorithm are also discussed.
PURPOSE - To determine the tolerance of 0.021-inch and 0.027-inch microcatheters to power injection in an in vitro flow model.
MATERIALS AND METHODS - Twenty-four microcatheters (0.021-inch, n = 13; 0.027-inch, n = 11) were injected with iothalamate meglumine through a flow model with use of a power injector and high-pressure tubing. Catheters used included Rebar (0.021-inch, n = 4; 0.027-inch, n = 4), Transit (0.021-inch, n = 3; 0.027-inch, n = 3), Renegade (0.021-inch, n = 4; 0.027-inch, n = 4), and Renegade STC-18 (0.021-inch, n = 2) models. Through the 0.021-inch microcatheters, 5-second injections were performed at an initial rate of 0.7 mL/sec. Injection rates were increased by 0.5 mL/sec and the process was repeated until the pressure approached 1,000 psi or catheter breakage occurred. A similar process was repeated for the 0.027-inch catheters starting at a rate of 3.4 mL/sec.
RESULTS - The 0.021-inch catheters were injected 303 times and the 0.027-inch catheters were injected 210 times. Three catheter failures occurred, with all breaks occurring at pressures greater than manufacturer recommendations. The 0.027-inch catheters as a group tolerated significantly higher injection rates than the 0.021-inch catheters. Of the 0.021-inch catheters, the STC-18 also provided superior maximum flow and volume compared with the Renegade catheter. The Rebar catheter tolerated significantly lower maximum injection rates and volumes than the other 0.027-inch catheters.
CONCLUSIONS - The majority of microcatheters can be power-injected in vitro at pressures far greater than manufacturer recommendations. When fractures occur, they are near the hub of the catheter. Significantly greater rates of injection are possible through 0.027-inch catheters.
A new formulation of a tissue-equivalent polymer-gel dosimeter for the measurement of three-dimensional dose distributions of ionizing radiation has been developed. It is composed of aqueous gelatin infused with acrylamide and N, N'-methylene-bisacrylamide monomers, and made hypoxic by nitrogen saturation. Irradiation of the gel, referred to as BANG, causes localized polymerization of the monomers, which, in turn, reduces the transverse NMR relaxation times of water protons. The dose dependence of the NMR transverse relaxation rate, R2, is reproducible (less than 2% variation) and is linear up to about 8 Gy, with a slope of 0.25 s(-1)Gy(-1) at 1.5 T. Magnetic resonance imaging may be used to obtain accurate three-dimensional dose distributions with high spatial resolution. Since the radiation-induced polymers do not diffuse through the gelatin matrix, the dose distributions recorded by BANG gels are stable for long periods of time, and may be used to measure low-activity radioactive sources. Since the light-scattering properties of the polymerized regions are different from those of the clear, non-irradiated regions, the dose distributions are visible, and their optical densities are dependent on dose.
There is a need for microminiaturized cell-culture environments, i.e. NanoLiter BioReactors (NBRs), for growing and maintaining populations of up to several hundred cultured mammalian cells in volumes three orders of magnitude smaller than those contained in standard multi-well screening plates. These devices would enable the development of a new class of miniature, automated cell-based bioanalysis arrays for monitoring the immediate environment of multiple cell lines and assessing the effects of drug or toxin exposure. We fabricated NBR prototypes, each of which incorporates a culture chamber, inlet and outlet ports, and connecting microfluidic conduits. The fluidic components were molded in polydimethylsiloxane (PDMS) using soft-lithography techniques, and sealed via plasma activation against a glass slide, which served as the primary culture substrate in the NBR. The input and outlet ports were punched into the PDMS block, and enabled the supply and withdrawal of culture medium into/from the culture chamber (10-100 nL volume), as well as cell seeding. Because of the intrinsically high oxygen permeability of the PDMS material, no additional CO(2)/air supply was necessary. The developmental process for the NBR typically employed several iterations of the following steps: Conceptual design, mask generation, photolithography, soft lithography, and proof-of-concept culture assay. We have arrived at several intermediate designs. One is termed "circular NBR with a central post (CP-NBR)," another, "perfusion (grid) NBR (PG-NBR)," and a third version, "multitrap (cage) NBR (MT-NBR)," the last two providing total cell retention. Three cells lines were tested in detail: a fibroblast cell line, CHO cells, and hepatocytes. Prior to the culturing trials, extensive biocompatibility tests were performed on all materials to be employed in the NBR design. To delineate the effect of cell seeding density on cell viability and survival, we conducted separate plating experiments using standard culture protocols in well-plate dishes. In both experiments, PicoGreen assays were used to evaluate the extent of cell growth achieved in 1-5 days following the seeding. Low seeding densities resulted in the absence of cell proliferation for some cell lines because of the deficiency of cell-cell and extracellular matrix (ECM)-cell contacts. High viabilities were achieved in all designs. We conclude that an instrumented microfluidics-based NanoBioReactor (NBR) will represent a dramatic departure from the standard culture environment. The employment of NBRs for mammalian cell culture opens a new paradigm of cell biology, so far largely neglected in the literature.
Closure of the abdominal wall after trauma or major surgery may be difficult due to visceral edema or fascial weakness; thus, the risk of developing a ventral hernia (VH) is high. Commonly, these hernias are repaired using a prosthetic mesh. Complications following mesh repair can develop. We hypothesize that the type of prosthetic material affects outcome. This is a retrospective chart review of patients admitted from 1996 to 2002 undergoing VH (> or = 20 x 10 cm) repair with prosthetic mesh. Data collected included age, sex, and race. Patients were stratified by prosthetic material as follows: Gore-Tex (GR), Marlex + Gore-Tex (MG), Marlex (MR), and Marlex + Vicryl (MV). For the purpose of clinical analysis, the groups were collapsed into subgroups: Gore-Tex exposure (GT) or non-Gore-Tex exposure (NG). Outcome measures were hernia recurrence (HR), wound infection (WI), and fistula formation (FF). Statistical analysis utilized chi2 test and Fisher's exact test. There were 55 VH repairs in 37 patients. The mean age was 43.9 (+/- 16.3), males out-numbered females 22 (59.5%) to 15 (40.5%). The majority of the patients were Caucasian (29; 78.4%). There were 30 trauma patients (81.1%), and 7 general surgery patients (18.9%). The HR for the study (n = 55) was 20 (36.4%), the WI was 17 (30.9%), and the FF was 3 (5.5%). GR group (6; 66.7%) had a significant higher wound IF rate than MR group (8; 26.7%) (Chi P = 0.02, Fisher P = 0.047). All other group comparisons (HR, WI, and FF) were N.S. The Gore-Tex versus non-Gore-Tex subgroup comparison results were as follows: GT (n = 18) had a WI 8 (44.4%), HR 6 (33.3%), and FF 0 (0%). NG (n = 37) had a WI 9 (24.3%), HR 14 (37.8%), and a FF 3 (8.1%). There was a trend toward a higher wound infection in the GT versus NG, but it did not reach statistical significance. We conclude that 1) the wound infection rate was higher in the Gore-Tex versus the Marlex group (Chi P = 0.02, Fisher P = 0.047). Wound infection in the presence of Gore-Tex usually mandates the removal of the mesh resulting in a hernia recurrence. 2) There was a trend toward a higher wound infection in the GT (44.4%) versus NG (24.3%), but it did not reach statistical significance.
Hemodialysis vascular access failure represents a major source of morbidity and mortality in chronic hemodialysis (CHD) patients. Serial vascular access blood flow (VABF) measurements are being used as a screening method at an increasing rate. There are limited data on the changes in VABF throughout the hemodialysis session, which may potentially affect the validity of VABF measurement. This study is performed to evaluate the trend in VABF during a given hemodialysis session by serial VABF measurements, along with potential factors that may affect VABF. Thirty-two CHD patients had serial VABF measurements performed during a hemodialysis session. Each patient had three serial VABF measurements during a hemodiaysis treatment (within 30, 90, and 150 minutes from the start of hemodialysis). Mean arterial blood pressure (MAP), ultrafiltration rate, and patient symptoms were recorded simultaneously. The mean VABF was 1,344 +/- 486 mL/min within 30 minutes of hemodialysis and decreased to 1,308 +/- 532 and 1,250 +/- 552 mL/min after 90 and 150 minutes, respectively. This trend was statistically significant (P = 0.03). There was a strong correlation between VABF measurements and MAP, which was more pronounced after 90 minutes of initiation of hemodialysis (r = 0.68; P < 0.001). Using multivariate analysis, it can be predicted that after 90 minutes of hemodialysis, each 10% decrease in MAP would result in an expected decrease of 8% in VABF. There was no effect of type of vascular access, baseline VABF, or amount of ultrafiltration on VABF changes. In conclusion, VABF measurements can be performed up to 2 to 2(1/2) hours from the start of hemodialysis in the majority of patients. The major determinant of VABF changes is MAP. In a subset of patients with a decrease MAP greater than 15%, it is advisable to perform VABF measurement either at the first 90 minutes of hemodialysis or postpone it to another treatment session, when MAP is more stable.