Sagar K. Gupta

Capture of circulating tumor cells using photoacoustic flowmetry and two phase flow

Melanoma is the deadliest form of skin cancer, yet current diagnostic methods are unable to detect early onset of metastatic disease. Patients must wait until macroscopic secondary tumors form before malignancy can be diagnosed and treatment prescribed. Detection of cells that have broken off the original tumor and travel through the blood or lymph system can provide data for diagnosing and monitoring metastatic disease. By irradiating enriched blood samples spiked with cultured melanoma cells with nanosecond duration laser light, we induced photoacoustic responses in the pigmented cells. Thus, we can detect and enumerate melanoma cells in blood samples to demonstrate a paradigm for a photoacoustic flow cytometer. Furthermore, we capture the melanoma cells using microfluidic two phase flow, a technique that separates a continuous flow into alternating microslugs of air and blood cell suspension. Each slug of blood cells is tested for the presence of melanoma. Slugs that are positive for melanoma, indicated by photoacoustic waves, are separated from the cytometer for further purification and isolation of the melanoma cell. In this paper, we evaluate the two phase photoacoustic flow cytometer for its ability to detect and capture metastatic melanoma cells in blood.

Detection of melanoma cells in vitro using an optical detector of photoacoustic waves.

Circulating tumor cells have been shown to correlate positively with metastatic disease state in patients with advanced cancer. We have demonstrated the ability to detect melanoma cells in a flow system by generating and detecting photoacoustic waves in melanoma cells. This method is similar to flow cytometry, although using photoacoustics rather than fluorescence. Previously, we used piezoelectric films as our acoustic sensors. However, such films have indicated false‐positive signals due to unwanted direct interactions between photons from the high laser fluence in the flow system and the film itself. We have adapted an optical detection scheme that obviates the need for piezoelectric films.

Photothermal therapy mediated by gum Arabic-conjugated gold nanoparticles suppresses liver preneoplastic lesions in mice

This study validates the utility of Gum Arabic-conjugated gold nanoparticles (GA-AuNPs) and laser to induce photothermal inhibition of hepatocarcinogenesis, via employing a diethylnitrosamine (DEN)-mediated hepatocellular carcinoma model. This work included both of in vitro and in vivo studies; to investigate the GA-AuNPs cytotoxicity and phototoxicity in hepatic cell line; to delineate the GA-AuNPs therapeutic efficiency in DEN-induced preneoplastic lesions (PNLs) in the liver of Balb-C mice. The therapeutic effects of GA-AuNPs on the mediators of apoptosis, inflammation, and tumor initiation, as well as the histopathological changes in preneoplastic liver have been investigated. Our results infer that GA-AuNPs in combination with laser irradiation led to a significant reduction in the cell viability and in histone deacetylase activity in hepatocarcinoma HepG2 cells. In chemically-induced PNLs mice model our results have demonstrated that GA-AuNPs, with or without laser irradiation, induced cancer cell apoptosis through the activation of death receptors DR5 and caspase-3 and inhibited both of the PNLs incidence and the initiation marker (placental glutathione S-transferase; GST-P). The laser-stimulated GA-AuNPs significantly reduced the tumor necrosis factor-α levels. In summary, GA-AuNPs with laser treatment inhibited liver PNLs via the induction of the extrinsic apoptosis pathway and the inhibition of inflammation.

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed secondary tumors(1,2,3). CTCs are indicators of metastatic disease and their detection in blood samples may be used to diagnose cancer and monitor a patients response to therapy. Since CTCs are rare, comprising about one tumor cell among billions of normal blood cells in advanced cancer patients, their detection and enumeration is a difficult task. We exploit the presence of pigment in most melanoma cells to generate photoacoustic, or laser induced ultrasonic waves in a custom flow cytometer for detection of circulating melanoma cells (CMCs)(4,5). This process entails separating a whole blood sample using centrifugation and obtaining the white blood cell layer. If present in whole blood, CMCs will separate with the white blood cells due to similar density. These cells are resuspended in phosphate buffered saline (PBS) and introduced into the flowmeter. Rather than a continuous flow of the blood cell suspension, we induced two phase flow in order to capture these cells for further study. In two phase flow, two immiscible liquids in a microfluidic system meet at a junction and form alternating slugs of liquid(6,7). PBS suspended white blood cells and air form microliter slugs that are sequentially irradiated with laser light. The addition of a surfactant to the liquid phase allows uniform slug formation and the user can create different sized slugs by altering the flow rates of the two phases. Slugs of air and slugs of PBS with white blood cells contain no light absorbers and hence, do not produce photoacoustic waves. However, slugs of white blood cells that contain even single CMCs absorb laser light and produce high frequency acoustic waves. These slugs that generate photoacoustic waves are sequestered and collected for cytochemical staining for verification of CMCs.

Photoacoustic detection of induced melanoma in vitro using a mouse model

Metastasis is a life threatening complex physiological phenomenon that involves the movement of cancer cells from one organ to another by means of blood and lymph. An understanding about metastasis is extremely important to device diagnostic systems to detect and monitor its spread within the body. For the first time we report rapid photoacoustic detection of the induced metastatic melanoma in mice in vitro using photoacoustic flowmetry. A new photoacoustic flow system is developed, that employs photoacoustic excitation coupled with an ultrasound transducer capable of determining the presence of individual, induced mouse melanoma cells (B16/F10) within the circulating system in vitro. Tumor was induced in mice by injecting mouse melanoma cells through tail vein into the C57BL/6 mice. A luciferase based in vivo bioluminescence imaging is performed to confirm the tumor load and multiple metastases in the tumor-induced mice. 1ml of blood obtained through cardiac puncture of the induced metastasized mice was treated to lyse the red blood cells (RBC) and enriched, leaving the induced melanoma in the peripheral blood mononuclear suspension (PBMC). A photoacoustic flowsystem coupled with an ultrasound transducer is used to detect the individual circulating metastatic melanoma cells from the enriched cell suspension.

Isolation of circulating tumor cells using photoacoustic flowmetry and two phase flow

Melanoma is the deadliest form of skin cancer, yet current diagnostic methods are inadequately sensitive. Patients must wait until secondary tumors form before malignancy can be diagnosed and treatment prescribed. Detection of cells that have broken off the original tumor and flow through the blood or lymph system can provide data for diagnosing and monitoring cancer. Our group utilizes the photoacoustic effect to detect metastatic melanoma cells, which contain the pigmented granule melanin. As a rapid laser pulse irradiates melanoma, the melanin undergoes thermo-elastic expansion and ultimately creates a photoacoustic wave. Thus, melanoma patients blood samples can be enriched, leaving the melanoma in a white blood cell (WBC) suspension. Irradiated melanoma cells produce photoacoustic waves, which are detected with a piezoelectric transducer, while the optically transparent WBCs create no signals. Here we report an isolation scheme utilizing two-phase flow to separate detected melanoma from the suspension. By introducing two immiscible fluids through a t-junction into one flow path, the analytes are compartmentalized. Therefore, the slug in which the melanoma cell is located can be identified and extracted from the system. Two-phase immiscible flow is a label free technique, and could be used for other types of pathological analytes.

Photoacoustic detection of gold nanorods tagged prostate cancer cells in vitro

Our recent efforts are to develop a system for the detection of extremely low number of prostate cancer cells tagged to gold nanorods. Such a system provides an attractive platform to detect the early metastasis. By monitoring the metastatic prostate cancer cells, the physicians are provided with more time to act and design the treatment and also provide better hope for the prostate cancer patients. We developed an optical flowmetry system which employs photoacoustic excitation coupled with an optical transducer capable of determining the presence of cells within the circulating system in vitro.The particles were tagged to gold nanoparticles at Dr.Kattis lab. This provided the required optical contrast to detect the individual prostate cancer cells. Detection trials resulted in a detection threshold of the order of a couple of individual cells in the detection volume, thus validating the effectiveness of the proposed mechanism.

A Comprehensive Review of the Present and Future Antibiotic Susceptibility Testing (AST) Systems

Tools and instruments available in the clinical microbiology labs for analysis of patient samples and diagnosis are constantly evolving. The main impetus behind this is to decrease the overall time taken to obtain the results from the instruments, enhance the ease of sample processing, increasing the sample turn-around time with the ultimate goal of earlier patient treatment and better recovery rates. This is especially true in the case of antibiotic susceptibility testing (AST), where every hour saved in obtaining the results leading to an earlier switch to targeted antibiotic therapy will have a direct influence on improving clinical outcomes. Reduction in the time to obtain AST results reduces the duration of use of broad-spectrum antibiotics, which in turn decreases the emergence of antibiotic resistance among bacteria. Many of the traditional methods available for AST are labor intensive and slow despite being precise in obtaining results. Thus, there is a trend towards development and use of automated diagnostic devices which are rapid and easy to use. This review article provides a detailed summary of traditional AST methods, currently used automated methods, and focuses on some of the promising emerging and future technologies in the field of rapid antibiotic susceptibility profiling.