Norman Barsalou

Detection of gold-nanorod targeted pathogens using optical and piezoelectric optoacoustic sensors: comparative study

We demonstrated the ability to detect surface antigens, associated with pathogens, utilizing laser optoacoustic spectroscopy with antibody-coupled gold nanorods (GNR) as a contrast agent specifically targeted to the antigen of interest. The sensitivity of the technique has been assessed by determining the minimum detectable concentration of a surface antigen from biological samples. We compared the sensitivity and applicability of two different methods for detecting optoacoustic responses, using either optical or piezoelectric sensors. Biological samples were adsorbed to the inside walls of detachable, flat-bottomed plastic micro-wells, and then probed with appropriate antibodies conjugated with gold nanorods. If the target antigens were present, the antibody-nanoparticle conjugates were bound, while any nonadsorbed nanoparticles were washed out of the wells. Optoacoustic signals were generated from the bound nanorods using a pulsed pump laser at wavelengths corresponding to one of the peak absorptions of the nanorods. Optoacoustic responses were obtained from the samples using both detection modalities. The sensitivity, suitability, ease of use of each method were assessed and compared. Initial results indicate that optical detection gives comparable sensitivity as the piezoelectric method, and further enhancement of the detection sensitivity is possible for both methods. An advantage of the piezoelectric detection method is that it may be implemented in a more compact assembly, compared to the optical method, however, the optical method may be less sensitive to external electromagnetic and acoustic noise. Further evaluation will be required to refine these measurements. The results with both methods indicate that the use of antibody-targeted nanorod contrast agents, with laser-optoacoustic detection, is a promising technology for the development of rapid in vitro diagnostic tests.

OPTOACOUSTIC SENSING OF OCULAR BACTERIAL ANTIGEN USING TARGETED GOLD NANORODS

Bacterial contamination can be detected using a minimally invasive optical method, based on laser-induced optoacoustic spectroscopy, to probe for specific antigens associated with a specific infectious agent. As a model system, we have used a surface antigen (Ag), isolated from Chlamydia trachomatis, and a complementary antibody (Ab). A preparation of 0.2 mg/ml of monoclonal Ab specific to the C. trachomatis surface Ag was conjugated to gold nanorods using standard commercial reagents, in order to produce a targeted contrast agent with a strong optoacoustic signal. The C. trachomatis Ag was absorbed in standard plastic microwells, and the binding of the complementary Ab-nanorod conjugate was tested in an immunoaffinity assay. Optoacoustic signals were elicited from the bound nanorods, using an optical parametric oscillator (OPO) laser system as the optical pump. The wavelength tuneability of the OPO optimized the spectroscopic measurement by exciting the nanorods at their optical absorption maxima. Optoacoustic responses were measured in the microwells using a probe beam deflection technique. Immunoaffinity assays were performed on several dilutions of purified C. trachomatis antigen ranging from 50 μg/ml to 1 pg/ml, in order to determine the detection limit for the optoacoustic-based assay. Only when the antigen was present, and the complementary Ab-NR reagent was introduced into the microwell, was an enhanced optoacoustic signal obtained, which indicated specific binding of the Ab-NR complex. The limit of detection with the current system design is between 1 and 5 pg/ml of bacterial Ag.

Optoacoustic sensor for nanoparticle linked immunosorbent assay (NanoLISA)

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.

Investigational detection of pharmacological agents in the eye using photoacoustic spectroscopy

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.

Investigation of photoacoustic spectroscopy for biomolecular detection

We are developing a non- or minimally-invasive method for detecting and measuring specific drugs and biomolecules in vivo using photoacoustic spectroscopy (PAS). This pilot study investigated the feasibility of detecting the concentration of certain drugs in the vitreous or aqueous of the eye. As a prototype for using PAS for molecular detection in vivo, the technique was applied to the detection in a surrogate eye, of drugs with known optical spectrum such as Trypan Blue, Rose Bengal, and Amphotericin B (AB), at concentrations as low as 1 μg/ml. Chopped CW, or short pulse, Q-switch lasers, were used as pumping sources to generate ultrasonic photoacoustic signals in an ocular phantom containing the drug solutions. In addition to an ultrasonic hydrophone, the photothermal deflection technique (PhDT), a non-contact optical method with high sensitivity and fast response, were used to record the photoacoustic signals. The data from both detectors were compared over a range of drug concentrations. The photoacoustic signal generated from the retina was used as a reference, to measure the attenuation of light through drug solutions of different concentrations in the ocular phantom. The results indicated that photoacoustic spectroscopy is feasible in ocular phantoms incorporating ex vivo ocular tissue. The signals recorded using PAS were to be found to be linearly dependent on drug concentration, as predicted by theory. The photoacoustic method was found to be sensitive to drug concentrations as low as 1 μg/ml, a clinically relevant concentration for many drugs. Future work will be directed at adapting this method for in vivo measurement, and enhancing its sensitivity by using a tunable laser as the pump source.

Nano-LISA for in vitro diagnostic applications

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.

Optoacoustic imaging: application to the detection of foreign bodies

Detection of non-radio-opaque foreign bodies can be difficult. Current imaging modalities employed for detection of foreign bodies include: X-ray computed tomography, magnetic resonance, and ultrasound. Successful diagnosis of the presence of foreign bodies is variable because of the difficulty of differentiating them from soft tissue, gas, and bone. We are applying laser-induced optoacoustic imaging to the detection of foreign bodies. Tissue-simulating phantoms containing various common foreign bodies have been constructed. Images of these phantoms were generated by two laser-based optoacoustic methods utilizing different detection modalities. A pre-commercial imager developed by Seno Medical Instruments (San Antonio), incorporated an ultrasound transducer to detect induced optoacoustic responses, while a laboratory-built imaging system utilized an optical probe beam deflection technique (PBDT) to detect the optoacoustic responses. The laboratory-built unit also included an optical parametric oscillator as the pump, providing tunable wavelength output to optimize the optoacoustic measurements by probing the foreign bodies at their maximum optical absorption. Results to date have been encouraging; both methodologies have allowed us to reconstruct successfully the image of foreign-body containing phantoms. In preliminary work the PBDT approach appeared to produce higher resolution than did the ultrasound detector, possibly because PBDT is not constrained by the lower bandwidth limit imposed on the ultrasound transducer necessary to increase imaging depth. During the research in progress, we will compare the optoacoustic images to those generated by MRI, CT, and ultrasound, and continue to improve the resolution of the technique by using multiple detection sensors, and to improve image contrast by scanning foreign bodies over a range of wavelengths.

Optoacoustic detection of viral antigens using targeted gold nanorods

We are detecting antigens (Ag), isolated from infectious organisms, utilizing laser optoacoustic spectroscopy and antibody-coupled gold nanorod (NR) contrast agents specifically targeted to the antigen of interest. We have detected, in clinical ocular samples, both Herpes Simplex Virus Type 1 and 2 (HSV-1 and HSV-2) . A monoclonal antibody (Ab) specific to both HSV-1 and HSV-2 was conjugated to gold nanorods to produce a targeted contrast agent with a strong optoacoustic signal. Elutions obtained from patient corneal swabs were adsorbed in standard plastic micro-wells. An immunoaffinity reaction was then performed with the functionalized gold nanorods, and the results were probed with an OPO laser, emitting wavelengths at the peak absorptions of the nanorods. Positive optoacoustic responses were obtained from samples containing authentic (microbiologically confirmed) HSV-1 and HSV-2. To obtain an estimate of the sensitivity of the technique, serial dilutions from 1 mg/ml to 1 pg/ml of a C. trachomatis surface Ag were prepared, and were probed with a monoclonal Ab, specific to the C. trachomatis surface Ag, conjugated to gold nanorods. An optoacoustic response was obtained, proportional to the concentration of antigen, and with a limit of detection of about 5 pg/ml. The optoacoustic signals generated from micro-wells containing albumin or saline were similar to those from blank wells. The potential benefit of this method is identify viral agents more rapidly than with existing techniques. In addition, the sensitivity of the assay is comparable or superior to existing colorimetric- or fluorometric-linked immunoaffinity assays.

N-acetylcysteine and acute retinal laser lesions in the colubrid snake eye

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.