We developed an optoacoustic biosensor intended for the detection of bloodborne microorganisms using
immunoaffinity reactions of antibody-coupled gold nanorods as contrast agents specifically targeted to the antigen
of interest. Optoacoustic responses generated by the samples are detected using a wide band ultrasonic transducer.
The sensitivity of the technique has been assessed by determining minimally detectable optical density which
corresponds to the minimum detectable concentration of the target viral surface antigens. Both ionic solutions and
gold nanorods served as the contrast agent generating the optoacoustic response. The sensitivity of Nano-LISA is at
least OD=10-6 which allows reliable detection of 1 pg/ml (depending on the commercial antibodies that are used).
Adequate detection sensitivity, as well as lack of non-specific cross-reaction between antigens favors NanoLISA as
a viable technology for biosensor development.
We previously reported the detection of bacterial antigen with immunoaffinity reactions using laser
optoacoustic spectroscopy and antibody-coupled gold nanorods (Ab-NR) as a contrast agent specifically
targeted to the antigen of interest. The Nano-LISA (Nanoparticle Linked Immunosorbent Assay) method
has been adapted to detect three very common blood-borne viral infectious agents, i.e. human T-lymphotropic
virus (HTLV), human immunodeficiency virus (HIV) and hepatitis-B (Hep-B). These agents
were used in a model test panel to illustrate the performance of the Nano-LISA technique. A working
laboratory prototype of a Nano-LISA microplate reader-sensor was assembled and tested against the panel
containing specific antigens of each of the infectious viral agents. Optoacoustic (OA) responses generated
by the samples were detected using the probe beam deflection technique, an all-optical, non-contact
technique. A LabView graphical user interface was developed for control of the instrument and real-time
display of the test results. The detection limit of Nano-LISA is at least 1 ng/ml of viral antigen, and can
reach 10 pg/ml, depending on the binding affinity of the specific detection antibody used to synthesize the
Ab-NR. The method has sufficient specificity, i.e. the detection reagents do not cross-react with noncomplementary
antigens. Thus, the OA microplate reader, incorporating NanoLISA, has adequate
detection sensitivity and specificity for use in clinical in vitro diagnostic testing.
Acute (1-hr, 6-hr) and longer term (24-hr) effects of laser injury on retinal function and cellular responses have been studied in the Great Plains rat snake, Elaphe guttata emoryi. This animal is of interest for vision research because its eye has an all-cone retina. A linear array of 5 thermal lesions was placed in the retina of anesthetized animals, near the area centralis, using a Nd:VO4 laser (532 nm), that delivered 50 mW per 10-msec pulse. Retinal function was assessed with the pattern electroretinogram (PERG), recorded before and after the placement of the lesions. PERGs were elicited with counterphased square-wave gratings, and were analyzed by Fourier analysis. The fate of lesioned cells was assessed by immunohistological staining for the transcription factor, NF-&kgr;B (which is activated by ionizing and nonionizing radiation), as well as for the apoptosis marker, caspase-9. The normal snake PERG had the maximum, real amplitude frequency component, determined by Fourier analysis, at the reversal frequency of the grating (i.e. shifts/sec). In the hour following the lesion-producing laser exposures, the PERG response exhibited frequency doubling, i.e. a new response waveform appeared at twice the reversal frequency. By 24-hr post exposure, many lesioned photoreceptors
stained positively for both NF-&kgr;B and caspase 9. Because the PERG largely reflects retinal ganglion cell activity, the appearance of frequency doubling in the PERG suggests that complementary (push-pull) inputs to ganglion cells are disrupted by the laser lesions. The immunohistological results indicate that activation of NF- B is not necessarily associated with photoreceptor survival after a laser injury.
This research reports progress in our earlier investigation of detecting specific drug diffusion into eye tissue
using photoacoustic spectroscopy (PAS). A key improvement to the technique is using short pulse tunable
laser source to stimulate the photoacoustic effect in tissue. An optical parametric oscillator (OPO) laser
system was used as a pumping source to generate ultrasonic photoacoustic signals and employed to scan
through different wavelengths with 0.1nm wavelength resolution to determine spectra of different drug
solutions in an ocular phantom. The short pulse duration (5-10ns) of the OPO laser has significantly
increased the photoacoustic efficiency conversion, and the ability to tune its output from 210nm to1800nm
has provided a wide selection range that is useful for optimizing spectroscopic studies. PAS spectra of
different solutions of molecules, such as Trypan Blue, Rose Bengal, Indocyanine Green (ICG), and
Amphotericin B (AB), at concentrations as low as 1 &mgr;g/ml, were constructed and compared to their actual
optical absorption spectra. Ultrasonic hydrophone and photothermal deflection technique (PhDT), a noncontact
optical method, were both used to record the photoacoustic signals, and compared in terms of sensitivity and applicability to record signals from the ocular tissue-bearing phantom. The results show good agreement between the optical and photoacoustic spectra, which supports moving to an in vivo application of recording the PAS responses from the eye. Future work will be directed at adapting this method for in vivo measurements, as well as improve the data acquisition system for faster PAS signal analysis.
This study examined the role of oxidative stress and the effect of a single dose treatment with N-Acetylcysteine (NAC) on the temporal development of acute laser-induced retinal injury. We used the snake eye/Scanning Laser Ophthalmoscope (SLO) model, an in vivo, non-invasive ocular imaging technique, which has the ability to image cellular retinal detail and allows for studying morphological changes of retinal injury over time. For this study 12 corn-snakes (Elaphe g. guttata) received 5 laser exposures per eye, followed by either a single dose of the antioxidant NAC (150mg/kg, IP in sterile saline) or placebo. Laser exposures were made with a Nd: VO4 DPSS, 532nm laser, coaxially aligned to the SLO. Shuttered pulses were 20msec x 50 mW; 1mJ each. Retinal images were taken using a Rodenstock cSLO and were digitally recorded at 1, 6, 24-hrs, and at 3-wks post-exposure. Lesions were assessed by two raters blind to the conditions of the study yielding measures of damaged area and counts of missing or damaged photoreceptors. Treated eyes showed a significant beneficial effect overall, and these results suggest that oxidative stress plays a role in laser-induced retinal injury. The use of NAC or a similar antioxidant shows promise as a therapeutic tool.