Standing Member, NIH Study Section - Instrumentation and Systems Development (ISD) (2013 - 2017)
Co-Director, Section of Laparoscopic and Minimally Invasive Urologic Surgery (2006 - Present)
Chief of Urology, VA Palo Alto Health Care System (2006 - Present)
Honors & Awards
Research Scholar, American Foundation for Urologic Disease (2003-05)
Diversity Faculty Research Award, Stanford (2007)
Translational Science Award, Stanford Comprehensive Cancer Center (2007)
Faculty Fellows Leadership Program, Stanford (2008)
First Place, AUA Foundation Young Investigator Research Forum (2009)
Invited Speaker, Gordon Research Conferences - Bioanalytical Sensors (2010)
Scholar, AUA/CUA International Exchange Program (2012)
Board Certification, American Board of Urology (2008)
Fellowship, University of California, Los Angeles, Endourology / Minimally Invasive Surgery (2006)
Residency, University of California, Los Angeles, Urology (2003)
Internship, University of California, Los Angeles, Surgery (1998)
M.D., Stanford, Medicine (1997)
A.B., Harvard, Biology (1993)
Current Research and Scholarly Interests
Minimally Invasive Surgery (Endoscopic, Laparoscopic, and Robotic-Assisted)
Urinary Tract Infections
A Biochip for Rapid Diagnosis of Complicated Urinary Tract Infection
The purpose of this proposal is clinical validation of an electrochemical biochip for rapid pathogen identification and antibiotic susceptibility determination.
Stanford is currently not accepting patients for this trial.
Fiber-Optic Confocal Microscopy of the Urinary Tract Histopathology
The goal of the study is to develop a novel approach to obtain real time optical biopsy of urinary tract pathology (e.g., bladder tumors) during urinary tract endoscopy using a novel fibered confocal microscope.
- Independent Studies (5)
Postdoctoral Faculty Sponsor
Graduate and Fellowship Programs
Deep Sequencing of Urinary RNAs for Bladder Cancer Molecular Diagnostics.
Clinical cancer research : an official journal of the American Association for Cancer Research
The majority of bladder cancer patients present with localized disease and are managed by transurethral resection. However, the high rate of recurrence necessitates lifetime cystoscopic surveillance. Developing a sensitive and specific urine-based test would significantly improve bladder cancer screening, detection, and surveillance.RNA-seq was used for biomarker discovery to directly assess the gene expression profile of exfoliated urothelial cells in urine derived from bladder cancer patients (n=13) and controls (n=10). Eight bladder cancer specific and 3 reference genes identified by RNA-seq were quantitated by qPCR in a training cohort of 102 urine samples. A diagnostic model based on the training cohort was constructed using multiple logistic regression. The model was further validated in an independent cohort of 101 urines.418 genes were found to be differentially expressed between bladder cancer and controls. Validation of a subset of these genes was used to construct an equation for computing a probability of bladder cancer score (PBC) based on expression of 3-markers (ROBO1, WNT5A, and CDC42BPB). Setting PBC=0.45 as the cutoff for a positive test, urine testing using the 3-marker panel had overall 88% sensitivity and 92% specificity in the training cohort. The accuracy of the 3-marker panel in the independent validation cohort yielded an area under the curve of 0.87 and overall 83% sensitivity and 89% specificity.Urine-based molecular diagnostics using this 3-marker signature could provide a valuable adjunct to cystoscopy and may lead to a reduction of unnecessary procedures for bladder cancer diagnosis.
View details for DOI 10.1158/1078-0432.CCR-16-2610
View details for PubMedID 28193625
New and developing diagnostic technologies for urinary tract infections.
Nature reviews. Urology
Timely and accurate identification and determination of the antimicrobial susceptibility of uropathogens is central to the management of UTIs. Urine dipsticks are fast and amenable to point-of-care testing, but do not have adequate diagnostic accuracy or provide microbiological diagnosis. Urine culture with antimicrobial susceptibility testing takes 2-3 days and requires a clinical laboratory. The common use of empirical antibiotics has contributed to the rise of multidrug-resistant organisms, reducing treatment options and increasing costs. In addition to improved antimicrobial stewardship and the development of new antimicrobials, novel diagnostics are needed for timely microbial identification and determination of antimicrobial susceptibilities. New diagnostic platforms, including nucleic acid tests and mass spectrometry, have been approved for clinical use and have improved the speed and accuracy of pathogen identification from primary cultures. Optimization for direct urine testing would reduce the time to diagnosis, yet these technologies do not provide comprehensive information on antimicrobial susceptibility. Emerging technologies including biosensors, microfluidics, and other integrated platforms could improve UTI diagnosis via direct pathogen detection from urine samples, rapid antimicrobial susceptibility testing, and point-of-care testing. Successful development and implementation of these technologies has the potential to usher in an era of precision medicine to improve patient care and public health.
View details for DOI 10.1038/nrurol.2017.20
View details for PubMedID 28248946
Oncologic Procedures Amenable to Fluorescence-guided Surgery.
Annals of surgery
Although fluorescence imaging is being applied to a wide range of cancers, it remains unclear which disease populations will benefit greatest. Therefore, we review the potential of this technology to improve outcomes in surgical oncology with attention to the various surgical procedures while exploring trial endpoints that may be optimal for each tumor type.For many tumors, primary treatment is surgical resection with negative margins, which corresponds to improved survival and a reduction in subsequent adjuvant therapies. Despite unfavorable effect on patient outcomes, margin positivity rate has not changed significantly over the years. Thus, patients often experience high rates of re-excision, radical resections, and overtreatment. However, fluorescence-guided surgery (FGS) has brought forth new light by allowing detection of subclinical disease not readily visible with the naked eye.We performed a systematic review of clinicatrials.gov using search terms "fluorescence," "image-guided surgery," and "near-infrared imaging" to identify trials utilizing FGS for those received on or before May 2016.fluorescence surgery for tumor debulking, wide local excision, whole-organ resection, and peritoneal metastases.fluorescence in situ hybridization, fluorescence imaging for lymph node mapping, nonmalignant lesions, nonsurgical purposes, or image guidance without fluorescence.Initial search produced 844 entries, which was narrowed down to 68 trials. Review of literature and clinical trials identified 3 primary resection methods for utilizing FGS: (1) debulking, (2) wide local excision, and (3) whole organ excision.The use of FGS as a surgical guide enhancement has the potential to improve survival and quality of life outcomes for patients. And, as the number of clinical trials rise each year, it is apparent that FGS has great potential for a broad range of clinical applications.
View details for DOI 10.1097/SLA.0000000000002127
View details for PubMedID 28045715
- Optical Biopsy of Bladder Cancer Using Crowd-Sourced Assessment. JAMA surgery 2016; 151 (1): 90-93
Endoscopic molecular imaging of human bladder cancer using a CD47 antibody.
Science translational medicine
2014; 6 (260): 260ra148-?
A combination of optical imaging technologies with cancer-specific molecular imaging agents is a potentially powerful strategy to improve cancer detection and enable image-guided surgery. Bladder cancer is primarily managed endoscopically by white light cystoscopy with suboptimal diagnostic accuracy. Emerging optical imaging technologies hold great potential for improved diagnostic accuracy but lack imaging agents for molecular specificity. Using fluorescently labeled CD47 antibody (anti-CD47) as molecular imaging agent, we demonstrated consistent identification of bladder cancer with clinical grade fluorescence imaging systems, confocal endomicroscopy, and blue light cystoscopy in fresh surgically removed human bladders. With blue light cystoscopy, the sensitivity and specificity for CD47-targeted imaging were 82.9 and 90.5%, respectively. We detected variants of bladder cancers, which are diagnostic challenges, including carcinoma in situ, residual carcinoma in tumor resection bed, recurrent carcinoma following prior intravesical immunotherapy with Bacillus Calmette-Guérin (BCG), and excluded cancer from benign but suspicious-appearing mucosa. CD47-targeted molecular imaging could improve diagnosis and resection thoroughness for bladder cancer.
View details for DOI 10.1126/scitranslmed.3009457
View details for PubMedID 25355698
- Hedgehog Signaling Restrains Bladder Cancer Progression by Eliciting Stromal Production of Urothelial Differentiation Factors CANCER CELL 2014; 26 (4): 521-533
Advances and challenges in biosensor-based diagnosis of infectious diseases
EXPERT REVIEW OF MOLECULAR DIAGNOSTICS
2014; 14 (2): 225-244
Rapid diagnosis of infectious diseases and timely initiation of appropriate treatment are critical determinants that promote optimal clinical outcomes and general public health. Conventional in vitro diagnostics for infectious diseases are time-consuming and require centralized laboratories, experienced personnel and bulky equipment. Recent advances in biosensor technologies have potential to deliver point-of-care diagnostics that match or surpass conventional standards in regards to time, accuracy and cost. Broadly classified as either label-free or labeled, modern biosensors exploit micro- and nanofabrication technologies and diverse sensing strategies including optical, electrical and mechanical transducers. Despite clinical need, translation of biosensors from research laboratories to clinical applications has remained limited to a few notable examples, such as the glucose sensor. Challenges to be overcome include sample preparation, matrix effects and system integration. We review the advances of biosensors for infectious disease diagnostics and discuss the critical challenges that need to be overcome in order to implement integrated diagnostic biosensors in real world settings.
View details for DOI 10.1586/14737159.2014.888313
View details for Web of Science ID 000335324700011
View details for PubMedID 24524681
Matrix-insensitive protein assays push the limits of biosensors in medicine
2009; 15 (11): 1327-U130
Advances in biosensor technologies for in vitro diagnostics have the potential to transform the practice of medicine. Despite considerable work in the biosensor field, there is still no general sensing platform that can be ubiquitously applied to detect the constellation of biomolecules in diverse clinical samples (for example, serum, urine, cell lysates or saliva) with high sensitivity and large linear dynamic range. A major limitation confounding other technologies is signal distortion that occurs in various matrices due to heterogeneity in ionic strength, pH, temperature and autofluorescence. Here we present a magnetic nanosensor technology that is matrix insensitive yet still capable of rapid, multiplex protein detection with resolution down to attomolar concentrations and extensive linear dynamic range. The matrix insensitivity of our platform to various media demonstrates that our magnetic nanosensor technology can be directly applied to a variety of settings such as molecular biology, clinical diagnostics and biodefense.
View details for DOI 10.1038/nm.2032
View details for Web of Science ID 000271543700023
View details for PubMedID 19820717
Long-range electrothermal fluid motion in microfluidic systems
INTERNATIONAL JOURNAL OF HEAT AND MASS TRANSFER
2016; 98: 341-349
AC electrothermal flow (ACEF) is the fluid motion created as a result of Joule heating induced temperature gradients. ACEF is capable of performing major microfluidic operations, such as pumping, mixing, concentration, separation and assay enhancement, and is effective in biological samples with a wide range of electrical conductivity. Here, we report long-range fluid motion induced by ACEF, which creates centimeter-scale vortices. The long-range fluid motion displays a strong voltage dependence and is suppressed in microchannels with a characteristic length below ~300 μm. An extended computational model of ACEF, which considers the effects of the density gradient and temperature-dependent parameters, is developed and compared experimentally by particle image velocimetry. The model captures the essence of ACEF in a wide range of channel dimensions and operating conditions. The combined experimental and computational study reveals the essential roles of buoyancy, temperature rise, and associated changes in material properties in the formation of the long-range fluid motion. Our results provide critical information for the design and modeling of ACEF based microfluidic systems toward various bioanalytical applications.
View details for DOI 10.1016/j.ijheatmasstransfer.2016.03.034
View details for Web of Science ID 000375360600032
- Real time diagnosis of bladder cancer with probe-based confocal laser endomicroscopy Proc. SPIE 7902, 79021V (2011); doi:10.1117/12.874243
- Hybrid electrokinetic manipulation in high-conductivity media Lab Chip (DOI: 10.1039/C1LC20054B)
- Editorial Comment. journal of urology 2016; 195 (6): 1703-?
A Microfiltration Device for Urogenital Schistosomiasis Diagnostics
2016; 11 (4)
Schistosomiasis is a parasitic disease affecting over 200 million people worldwide. This study reports the design and development of a microfiltration device for isolating schistosome eggs in urine for rapid diagnostics of urogenital schistosomiasis. The design of the device comprises a linear array of microfluidic traps to immobilize and separate schistosome eggs. Sequential loading of individual eggs is achieved autonomously by flow resistance, which facilitates observation and enumeration of samples with low-abundance targets. Computational fluid dynamics modeling and experimental characterization are performed to optimize the trapping performance. By optimizing the capture strategy, the trapping efficiency could be achieved at 100% with 300 μl/min and 83% with 3000 μl/min, and the filtration procedure could be finished within 10 min. The trapped eggs can be either recovered for downstream analysis or preserved in situ for whole-mount staining. On-chip phenotyping using confocal laser fluorescence microscopy identifies the microstructure of the trapped schistosome eggs. The device provides a novel microfluidic approach for trapping, counting and on-chip fluorescence characterization of urinal Schistosoma haematobium eggs for clinical and investigative application.
View details for DOI 10.1371/journal.pone.0154640
View details for Web of Science ID 000375211700119
View details for PubMedID 27124499
- Intraoperative Optical Biopsy during Robotic Assisted Radical Prostatectomy Using Confocal Endomicroscopy JOURNAL OF UROLOGY 2016; 195 (4): 1110-1117
- Multimodal 3D cancer-mimicking optical phantom BIOMEDICAL OPTICS EXPRESS 2016; 7 (2): 648-662
Registration of free-hand OCT daughter endoscopy to 3D organ reconstruction.
Biomedical optics express
2016; 7 (12): 4995–5009
Despite the trend to pair white light endoscopy with secondary image modalities for in vivo characterization of suspicious lesions, challenges remain to co-register such data. We present an algorithm to co-register two different optical imaging modalities as a mother-daughter endoscopy pair. Using white light cystoscopy (mother) and optical coherence tomography (OCT) (daughter) as an example, we developed the first forward-viewing OCT endoscope that fits in the working channel of flexible cystoscopes and demonstrated our algorithm's performance with optical phantom and clinical imaging data. The ability to register multimodal data opens opportunities for advanced analysis in cancer imaging applications.
View details for DOI 10.1364/BOE.7.004995
View details for PubMedID 28018720
A Multiplex Electrochemical Biosensor for Bloodstream Infection Diagnosis.
Journal of laboratory automation
Accurate and timely detection of bacterial pathogens will improve the clinical management of infections. Herein, we demonstrate an electrochemical biosensor that directly detects bacteria in human blood samples, resulting in the rapid diagnosis of a bloodstream infection. The multiplex biosensor detects the species-specific sequences of the 16S ribosomal RNA of bacteria for pathogen identification in physiological samples without preamplification. The analytical performance characteristics of the biosensor, including the limit of detection and probe cross-reactivity, are evaluated systematically. The feasibility of the biosensor for a diagnosis of a bloodstream infection is demonstrated by identifying bacterial clinical isolates spiked in whole blood and blood culture samples that were tested positive for bacteria. The electrochemical biosensor correctly identifies all the species in the samples with 100% concordance to microbiological analysis.
View details for DOI 10.1177/2211068216651232
View details for PubMedID 27226118
- Successful Translation of Fluorescence Navigation During Oncologic Surgery: A Consensus Report JOURNAL OF NUCLEAR MEDICINE 2016; 57 (1): 144-150
Ultra-thin elastomer membrane array wrinkling for bladder cancer diagnosis
IEEE 29th International Conference on Micro Electro Mechanical Systems (MEMS)
View details for DOI 10.1109/MEMSYS.2016.7421650
Multimodal 3D cancer-mimicking optical phantom.
Biomedical optics express
2016; 7 (2): 648-62
Three-dimensional (3D) organ-mimicking phantoms provide realistic imaging environments for testing various aspects of optical systems, including for evaluating new probe designs, characterizing the diagnostic potential of new technologies, and assessing novel image processing algorithms prior to validation in real tissue. We introduce and characterize the use of a new material, Dragon Skin (Smooth-On Inc.), and fabrication technique, air-brushing, for fabrication of a 3D phantom that mimics the appearance of a real organ under multiple imaging modalities. We demonstrate the utility of the material and technique by fabricating the first 3D, hollow bladder phantom with realistic normal and multi-stage pathology features suitable for endoscopic detection using the gold standard imaging technique, white light cystoscopy (WLC), as well as the complementary imaging modalities of optical coherence tomography and blue light cystoscopy, which are aimed at improving the sensitivity and specificity of WLC to bladder cancer detection. The flexibility of the material and technique used for phantom construction allowed for the representation of a wide range of diseased tissue states, ranging from inflammation (benign) to high-grade cancerous lesions. Such phantoms can serve as important tools for trainee education and evaluation of new endoscopic instrumentation.
View details for DOI 10.1364/BOE.7.000648
View details for PubMedID 26977369
- Editorial Comment. The Journal of urology 2016
AC Electrokinetics of Physiological Fluids for Biomedical Applications
2015; 20 (6): 611-620
Alternating current (AC) electrokinetics is a collection of processes for manipulating bulk fluid mass and embedded objects with AC electric fields. The ability of AC electrokinetics to implement the major microfluidic operations, such as pumping, mixing, concentration, and separation, makes it possible to develop integrated systems for clinical diagnostics in nontraditional health care settings. The high conductivity of physiological fluids presents new challenges and opportunities for AC electrokinetics-based diagnostic systems. In this review, AC electrokinetic phenomena in conductive physiological fluids are described followed by a review of the basic microfluidic operations and the recent biomedical applications of AC electrokinetics. The future prospects of AC electrokinetics for clinical diagnostics are presented.
View details for DOI 10.1177/2211068214560904
View details for Web of Science ID 000365429900001
View details for PubMedID 25487557
A Pilot Study of In Vivo Confocal Laser Endomicroscopy of Upper Tract Urothelial Carcinoma
JOURNAL OF ENDOUROLOGY
2015; 29 (12): 1418-1423
Tissue diagnosis of upper tract urothelial carcinoma (UTUC) is limited by variance in tumor sampling by standard ureteroscopic biopsy. Optical imaging technologies can potentially improve UTUC diagnosis, surveillance, and endoscopic treatment. We previously demonstrated in vivo optical biopsy of urothelial carcinoma of the bladder using confocal laser endomicroscopy (CLE). In this study, we evaluated a new 0.85-mm imaging probe in the upper urinary tract and demonstrated feasibility and compatibility with standard ureteroscopes to achieve in vivo optical biopsy of UTUC.Fourteen patients scheduled for ureteroscopy of suspected upper tract lesions or surveillance of UTUC were recruited. After intravenous (IV) administration of fluorescein, CLE was performed using a 0.85-mm-diameter imaging probe inserted through the working channel of standard ureteroscopes. Acquired confocal video sequences were reviewed and analyzed. A mosaicing algorithm was used to compile a series of images into a single larger composite image. Processed CLE images were compared with standard histopathologic analysis.Optical biopsy of the UTUC using CLE was effectively achieved during standard ureteroscopy. There were no adverse events related to IV fluorescein administration or image acquisition. Confocal imaging of UTUC showed characteristic features similar to urothelial carcinoma of the bladder, including papillary structure, fibrovascular stalks, and pleomorphism. Lamina propria in normal areas of the renal pelvis and ureter was also identified.We report an initial feasibility of CLE of UTUC. Pending further clinical investigation, CLE may become a useful adjunct to ureteroscopic biopsy, endoscopic ablation, and surveillance of UTUC.
View details for DOI 10.1089/end.2015.0523
View details for Web of Science ID 000366602600015
View details for PubMedID 26413927
Optical Biopsy of Peripheral Nerve Using Confocal Laser Endomicroscopy: A New Tool for Nerve Surgeons?
Archives of plastic surgery
2015; 42 (5): 626-629
Peripheral nerve injuries remain a challenge for reconstructive surgeons with many patients obtaining suboptimal results. Understanding the level of injury is imperative for successful repair. Current methods for distinguishing healthy from damaged nerve are time consuming and possess limited efficacy. Confocal laser endomicroscopy (CLE) is an emerging optical biopsy technology that enables dynamic, high resolution, sub-surface imaging of live tissue. Porcine sciatic nerve was either left undamaged or briefly clamped to simulate injury. Diluted fluorescein was applied topically to the nerve. CLE imaging was performed by direct contact of the probe with nerve tissue. Images representative of both damaged and undamaged nerve fibers were collected and compared to routine H&E histology. Optical biopsy of undamaged nerve revealed bands of longitudinal nerve fibers, distinct from surrounding adipose and connective tissue. When damaged, these bands appear truncated and terminate in blebs of opacity. H&E staining revealed similar features in damaged nerve fibers. These results prompt development of a protocol for imaging peripheral nerves intraoperatively. To this end, improving surgeons' ability to understand the level of injury through real-time imaging will allow for faster and more informed operative decisions than the current standard permits.
View details for DOI 10.5999/aps.2015.42.5.626
View details for PubMedID 26430636
Role of Narrow Band Imaging in Management of Urothelial Carcinoma
CURRENT UROLOGY REPORTS
2015; 16 (8)
Urothelial carcinoma of the bladder and upper tract is primarily diagnosed by white light endoscopy, which has well-known limitations that contribute to the increased risk of tumor recurrence and progression. Narrow band imaging (NBI) is an optical imaging technology that facilitates detection of tumor vasculature and differentiation of benign urothelium from neoplastic tissue. For urothelial carcinoma, NBI may be utilized in a variety of clinical settings, including office cystoscopy for initial identification and surveillance, transurethral resection for pathological diagnosis, and ureteroscopic management of upper tract lesions. Early evidence suggests that NBI increases the detection of urothelial carcinoma in the bladder and upper tract, including flat high-grade lesions such as carcinoma-in-situ that are a diagnostic challenge under white light. NBI also appears to improve the quality of transurethral resection and thereby reduce the frequency of tumor recurrence.
View details for DOI 10.1007/s11934-015-0527-5
View details for Web of Science ID 000357431600008
View details for PubMedID 26093973
Advances in Imaging Technologies in the Evaluation of High-Grade Bladder Cancer
UROLOGIC CLINICS OF NORTH AMERICA
2015; 42 (2): 147-?
Bladder cancer ranges from a low-grade variant to high-grade disease. Assessment for treatment depends on white light cystoscopy, however because of its limitations there is a need for improved visualization of flat, multifocal, high-grade, and muscle-invasive lesions. Photodynamic diagnosis and narrow-band imaging provide additional contrast enhancement of bladder tumors and have been shown to improve detection rates. Confocal laser endomicroscopy and optical coherence tomography enable real-time, high-resolution, subsurface tissue characterization with spatial resolutions similar to histology. Molecular imaging offers the potential for the combination of optical imaging technologies with cancer-specific molecular agents to improve the specificity of disease detection.
View details for DOI 10.1016/j.ucl.2015.01.001
View details for Web of Science ID 000354588200003
View details for PubMedID 25882557
- Editorial Comment. The Journal of urology 2015
Fiber-optic Confocal Laser Endomicroscopy of Small Renal Masses: Towards Real-time Optical Diagnostic Biopsy.
The Journal of urology
The incidental detection of small renal masses is on the rise. However, not all require aggressive treatments as up to 20% are benign and the majority of malignant tumors harbor indolent features. Improved preoperative diagnostics are needed to differentiate tumors requiring aggressive treatment from those more suitable for surveillance. We evaluated and compared confocal laser endomicroscopy (CLE) with standard histopathology in ex vivo human kidney tumors as a proof-of-principle towards diagnostic optical biopsy.Patients with solitary small renal masses scheduled for partial or radical nephrectomy were enrolled in the study. Two kidneys were infused with fluorescein via intraoperative intravenous injection, and 18 tumors were bathed ex vivo in dilute fluorescein prior to confocal imaging. A 2.6 mm CLE probe was used to image tumors and surrounding parenchyma from external and en face surfaces after specimen bisection. CLE images were compared to standard H&E analysis of corresponding areas.Ex vivo CLE imaging revealed normal renal structures that correlated well with histology. Tumor tissue was readily distinguishable from normal parenchyma, demonstrating features unique to benign and malignant tumor subtypes. Topical fluorescein administration provided more consistent CLE imaging than the intravenous route. Additionally, en face tumor imaging was superior to external imaging.We report the first feasibility study using CLE to evaluate small renal masses ex vivo and provide a preliminary atlas of images from various renal neoplasms with corresponding histology. These findings serve as an initial and promising step towards real-time, diagnostic optical biopsy of small renal masses.
View details for DOI 10.1016/j.juro.2015.07.115
View details for PubMedID 26321408
Rapid bladder cancer cell detection from clinical urine samples using an ultra-thin silicone membrane.
Early detection of initial onset, as well as recurrence, of cancer is paramount for improved patient prognosis and human health. Cancer screening is enhanced by rapid differentiation of cancerous from non-cancerous cells which employs the inherent differences in biophysical properties. Our preliminary testing demonstrates that cell-line derived bladder cancer cells deform our <30 nm silicone membrane within an hour and induce visually distinct wrinkle patterns while cell-line derived non-cancerous cells fail to induce these wrinkle patterns. Herein, we report a platform for the rapid detection of cancerous cells from human clinical urine samples. We performed a blinded study with cells extracted from the urine of human patients suspected to have bladder cancer alongside healthy controls. Wrinkle patterns were induced specifically by the five cancer patient samples within 12 hours and not by the healthy controls. These results were independently validated by the standard diagnostic techniques cystoscopy and cytology. Thus, our ultra-thin membrane approach for cancer diagnosis appears as accurate as standard diagnostic methods while vastly more rapid, less invasive, and requiring limited expertise.
View details for DOI 10.1039/c5an01616a
View details for PubMedID 26549051
Rapid Antimicrobial Susceptibility Testing with Electrokinetics Enhanced Biosensors for Diagnosis of Acute Bacterial Infections
ANNALS OF BIOMEDICAL ENGINEERING
2014; 42 (11): 2314-2321
Rapid pathogen detection and antimicrobial susceptibility testing (AST) are required in diagnosis of acute bacterial infections to determine the appropriate antibiotic treatment. Molecular approaches for AST are often based on the detection of known antibiotic resistance genes. Phenotypic culture analysis requires several days from sample collection to result reporting. Toward rapid diagnosis of bacterial infection in non-traditional healthcare settings, we have developed a rapid AST approach that combines phenotypic culture of bacterial pathogens in physiological samples and electrochemical sensing of bacterial 16S rRNA. The assay determines the susceptibility of pathogens by detecting bacterial growth under various antibiotic conditions. AC electrokinetic fluid motion and Joule heating induced temperature elevation are optimized to enhance the sensor signal and minimize the matrix effect, which improve the overall sensitivity of the assay. The electrokinetics enhanced biosensor directly detects the bacterial pathogens in blood culture without prior purification. Rapid determination of the antibiotic resistance profile of Escherichia coli clinical isolates is demonstrated.
View details for DOI 10.1007/s10439-014-1040-6
View details for Web of Science ID 000344091600011
View details for PubMedID 24889716
Confocal laser endomicroscopy of bladder and upper tract urothelial carcinoma: a new era of optical diagnosis?
Current urology reports
2014; 15 (9): 437-?
Urothelial carcinoma of the bladder and upper tract pose significant diagnostic and therapeutic challenges. White light endoscopy plays a central role in the management of urothelial carcinoma but has several well-recognized shortcomings. New optical imaging technologies may improve diagnostic accuracy, enhance local cancer control, and better stratify treatment options. Confocal laser endomicroscopy enables dynamic imaging of the cellular structures below the mucosal surface and holds promise in providing real time optical diagnosis and grading of urothelial carcinoma. A variety of imaging probes are available that are compatible with the full spectrum of cystoscopes and ureteroscopes. We review the underlying principles and technique of confocal laser endomicroscopy in the urinary tract, with emphasis on specific application towards urothelial carcinoma. While the available data are largely related to urothelial carcinoma of the bladder, the lessons learned are directly applicable to the upper tract, where the clinical needs are significant. Ongoing efforts to optimize this technology offer an exciting glimpse into future advances in optical imaging and intraoperative image guidance.
View details for DOI 10.1007/s11934-014-0437-y
View details for PubMedID 25002073
Emerging endoscopic imaging technologies for bladder cancer detection.
Current urology reports
2014; 15 (5): 406-?
Modern urologic endoscopy is the result of continuous innovations since the early nineteenth century. White-light cystoscopy is the primary strategy for identification, resection, and local staging of bladder cancer. While highly effective, white light cystoscopy has several well-recognized shortcomings. Recent advances in optical imaging technologies and device miniaturization hold the potential to improve bladder cancer diagnosis and resection. Photodynamic diagnosis and narrow band imaging are the first to enter the clinical arena. Confocal laser endomicroscopy, optical coherence tomography, Raman spectroscopy, UV autofluorescence, and others have shown promising clinical and pre-clinical feasibility. We review their mechanisms of action, highlight their respective advantages, and propose future directions.
View details for DOI 10.1007/s11934-014-0406-5
View details for PubMedID 24658832
Three-dimensional, distendable bladder phantom for optical coherence tomography and white light cystoscopy.
Journal of biomedical optics
2014; 19 (3): 36009-?
ABSTRACT. We describe a combination of fabrication techniques and a general process to construct a three-dimensional (3-D) phantom that mimics the size, macroscale structure, microscale surface topology, subsurface microstructure, optical properties, and functional characteristics of a cancerous bladder. The phantom also includes features that are recognizable in white light (i.e., the visual appearance of blood vessels), making it suitable to emulate the bladder for emerging white light+optical coherence tomography (OCT) cystoscopies and other endoscopic procedures of large, irregularly shaped organs. The fabrication process has broad applicability and can be generalized to OCT phantoms for other tissue types or phantoms for other imaging modalities. To this end, we also enumerate the nuances of applying known fabrication techniques (e.g., spin coating) to contexts (e.g., nonplanar, 3-D shapes) that are essential to establish their generalizability and limitations. We anticipate that this phantom will be immediately useful to evaluate innovative OCT systems and software being developed for longitudinal bladder surveillance and early cancer detection.
View details for DOI 10.1117/1.JBO.19.3.036009
View details for PubMedID 24623158
Intraoperative optical imaging and tissue interrogation during urologic surgery
CURRENT OPINION IN UROLOGY
2014; 24 (1): 66-74
To review optical imaging technologies in urologic surgery aimed to facilitate intraoperative imaging and tissue interrogation.Emerging new optical imaging technologies can be integrated in the operating room environment during minimally invasive and open surgery. These technologies include macroscopic fluorescence imaging that provides contrast enhancement between normal and diseased tissue and microscopic imaging that provides tissue characterization.Optical imaging technologies that have reached the clinical arena in urologic surgery were reviewed, including photodynamic diagnosis, near infrared fluorescence imaging, optical coherence tomography, and confocal laser endomicroscopy.
View details for DOI 10.1097/MOU.0000000000000010
View details for Web of Science ID 000327901400012
View details for PubMedID 24240512
Electrokinetic stringency control in self-assembled monolayer-based biosensors for multiplex urinary tract infection diagnosis
NANOMEDICINE-NANOTECHNOLOGY BIOLOGY AND MEDICINE
2014; 10 (1): 159-166
Rapid detection of bacterial pathogens is critical toward judicious management of infectious diseases. Herein, we demonstrate an in situ electrokinetic stringency control approach for a self-assembled monolayer-based electrochemical biosensor toward urinary tract infection diagnosis. The in situ electrokinetic stringency control technique generates Joule heating induced temperature rise and electrothermal fluid motion directly on the sensor to improve its performance for detecting bacterial 16S rRNA, a phylogenetic biomarker. The dependence of the hybridization efficiency reveals that in situ electrokinetic stringency control is capable of discriminating single-base mismatches. With electrokinetic stringency control, the background noise due to the matrix effects of clinical urine samples can be reduced by 60%. The applicability of the system is demonstrated by multiplex detection of three uropathogenic clinical isolates with similar 16S rRNA sequences. The results demonstrate that electrokinetic stringency control can significantly improve the signal-to-noise ratio of the biosensor for multiplex urinary tract infection diagnosis.Urinary tract infections remain a significant cause of mortality and morbidity as secondary conditions often related to chronic diseases or to immunosuppression. Rapid and sensitive identification of the causative organisms is critical in the appropriate management of this condition. These investigators demonstrate an in situ electrokinetic stringency control approach for a self-assembled monolayer-based electrochemical biosensor toward urinary tract infection diagnosis, establishing that such an approach significantly improves the biosensor's signal-to-noise ratio.
View details for DOI 10.1016/j.nano.2013.07.006
View details for Web of Science ID 000329103500017
View details for PubMedID 23891989
- A Universal Electrode Approach for Automated Electrochemical Molecular Analyses JOURNAL OF MICROELECTROMECHANICAL SYSTEMS 2013; 22 (5): 1126-1132
- Turning on the lights: new technologies in optical diagnostics and therapeutics. journal of urology 2013; 190 (2): 381-382
Interobserver Agreement of Confocal Laser Endomicroscopy for Bladder Cancer
JOURNAL OF ENDOUROLOGY
2013; 27 (5): 598-603
Emerging optical imaging technologies such as confocal laser endomicroscopy (CLE) hold promise in improving bladder cancer diagnosis. The purpose of this study was to determine the interobserver agreement of image interpretation using CLE for bladder cancer.Experienced CLE urologists (n=2), novice CLE urologists (n=6), pathologists (n=4), and nonclinical researchers (n=5) were recruited to participate in a 2-hour computer-based training consisting of a teaching and validation set of intraoperative white light cystoscopy (WLC) and CLE video sequences from patients undergoing transurethral resection of bladder tumor. Interobserver agreement was determined using the κ statistic.Of the 31 bladder regions analyzed, 19 were cancer and 12 were benign. For cancer diagnosis, experienced CLE urologists had substantial agreement for both CLE and WLC+CLE (90%, κ 0.80) compared with moderate agreement for WLC alone (74%, κ 0.46), while novice CLE urologists had moderate agreement for CLE (77%, κ 0.55), WLC (78%, κ 0.54), and WLC+CLE (80%, κ 0.59). Pathologists had substantial agreement for CLE (81%, κ 0.61), and nonclinical researchers had moderate agreement (77%, κ 0.49) in cancer diagnosis. For cancer grading, experienced CLE urologists had fair to moderate agreement for CLE (68%, κ 0.64), WLC (74%, κ 0.67), and WLC+CLE (53%, κ 0.33), as did novice CLE urologists for CLE (53%, κ 0.39), WLC (66%, κ 0.50), and WLC+CLE (61%, κ 0.49). Pathologists (65%, κ 0.55) and nonclinical researchers (61%, κ 0.56) both had moderate agreement for CLE in cancer grading.CLE is an adoptable technology for cancer diagnosis in novice CLE observers after a short training with moderate interobserver agreement and diagnostic accuracy similar to WLC alone. Experienced CLE observers may be capable of achieving substantial levels of agreement for cancer diagnosis that is higher than with WLC alone.
View details for DOI 10.1089/end.2012.0549
View details for Web of Science ID 000318552100014
Single Cell Antimicrobial Susceptibility Testing by Confined Microchannels and Electrokinetic Loading
2013; 85 (8): 3971-3976
Multidrug-resistant pathogens are an emerging global health problem. In addition to the need of developing new antibiotics in the pipeline, the ability to rapidly determine the antibiotic resistance profiles of bacteria represents one of the most crucial steps toward the management of infectious diseases and the prevention of multidrug-resistant pathogens. Here, we report a single cell antimicrobial susceptibility testing (AST) approach for rapid determination of the antibiotic resistance of bacterial pathogens. By confining individual bacteria in gas permeable microchannels with dimensions comparable to a single bacterium, the antibiotic resistance of the bacteria can be monitored in real-time at the single cell level. To facilitate the dynamic loading of the bacteria into the confined microchannels for observation, AC electrokinetics is demonstrated for capturing bacteria to defined locations in high-conductivity AST buffer. The electrokinetic technique achieves a loading efficiency of about 75% with a negligible effect on the bacterial growth rate. To optimize the protocol for single cell AST, the bacterial growth rate of individual bacteria under different antibiotic conditions has been determined systematically. The applicability of single cell AST is demonstrated by the rapid determination of the antimicrobial resistant profiles of uropathogenic clinical isolates in Mueller-Hinton media and in urine. The antibiotic resistance profiles of bacteria can be determined in less than 1 h compared to days in standard culture-based AST techniques.
View details for DOI 10.1021/ac4004248
View details for Web of Science ID 000317794800027
View details for PubMedID 23445209
An AC electrokinetics facilitated biosensor cassette for rapid pathogen identification
2013; 138 (13): 3660-3666
To develop a portable point-of-care system based on biosensors for common infectious diseases such as urinary tract infection, the sensing process needs to be implemented within an enclosed fluidic system. On chip sample preparation of clinical samples remains a significant obstacle to achieving robust sensor performance. Herein AC electrokinetics is applied in an electrochemical biosensor cassette to enhance molecular convection and hybridization efficiency through electrokinetics induced fluid motion and Joule heating induced temperature elevation. Using E. coli as an exemplary pathogen, we determined the optimal electrokinetic parameters for detecting bacterial 16S rRNA in the biosensor cassette based on the current output, signal-to-noise ratio, and limit of detection. In addition, a panel of six probe sets targeting common uropathogenic bacteria was demonstrated. The optimized parameters were also validated using patient-derived clinical urine samples. The effectiveness of electrokinetics for on chip sample preparation will facilitate the implementation of point-of-care diagnosis of urinary tract infection in the future.
View details for DOI 10.1039/c3an00259d
View details for Web of Science ID 000319876800012
View details for PubMedID 23626988
- Probe-based Confocal Laser Endomicroscopy of the Urinary Tract: The Technique JOVE-JOURNAL OF VISUALIZED EXPERIMENTS 2013
A Universal Electrode Approach for Automated Electrochemical Molecular Analyses.
Journal of microelectromechanical systems : a joint IEEE and ASME publication on microstructures, microactuators, microsensors, and microsystems
2013; 22 (5): 1126-1132
Transforming microfluidics-based biosensing systems from laboratory research into clinical reality remains an elusive goal despite decades of intensive research. A fundamental obstacle for the development of fully automated microfluidic diagnostic systems is the lack of an effective strategy for combining pumping, sample preparation, and detection modules into an integrated biosensing platform. Herein, we report a universal electrode approach, which incorporates DC electrolytic pumping, AC electrokinetic sample preparation, and self-assembled monolayer based electrochemical sensing on a single microfluidic platform, to automate complicated molecular analysis procedures that will enable biosensing applications in non-traditional healthcare settings. Using the universal electrode approach, major microfluidic operations required in molecular analyses, such as pumping, mixing, washing, and sensing can be performed in a single platform. We demonstrate the universal electrode platform for detecting bacterial 16S rRNA, a phylogenetic marker, toward rapid diagnostics of urinary tract infection. Since only electronic interfaces are required to operate the platform, the universal electrode approach represents an effective system integration strategy to realize the potential of microfluidics in molecular diagnostics at the point of care.
View details for DOI 10.1109/JMEMS.2013.2253545
View details for PubMedID 24860248
Probe-based confocal laser endomicroscopy of the urinary tract: the technique.
Journal of visualized experiments : JoVE
Probe-based confocal laser endomicroscopy (CLE) is an emerging optical imaging technology that enables real-time in vivo microscopy of mucosal surfaces during standard endoscopy. With applications currently in the respiratory and gastrointestinal tracts, CLE has also been explored in the urinary tract for bladder cancer diagnosis. Cellular morphology and tissue microarchitecture can be resolved with micron scale resolution in real time, in addition to dynamic imaging of the normal and pathological vasculature. The probe-based CLE system (Cellvizio, Mauna Kea Technologies, France) consists of a reusable fiberoptic imaging probe coupled to a 488 nm laser scanning unit. The imaging probe is inserted in the working channels of standard flexible and rigid endoscopes. An endoscope-based CLE system (Optiscan, Australia), in which the confocal endomicroscopy functionality is integrated onto the endoscope, is also used in the gastrointestinal tract. Given the larger scope diameter, however, application in the urinary tract is currently limited to ex vivo use. Confocal image acquisition is done through direct contact of the imaging probe with the target tissue and recorded as video sequences. As in the gastrointestinal tract, endomicroscopy of the urinary tract requires an exogenenous contrast agent-most commonly fluorescein, which can be administered intravenously or intravesically. Intravesical administration is a well-established method to introduce pharmacological agents locally with minimal systemic toxicity that is unique to the urinary tract. Fluorescein rapidly stains the extracellular matrix and has an established safety profile. Imaging probes of various diameters enable compatibility with different caliber endoscopes. To date, 1.4 and 2.6 mm probes have been evaluated with flexible and rigid cystoscopy. Recent availability of a < 1 mm imaging probe opens up the possibility of CLE in the upper urinary tract during ureteroscopy. Fluorescence cystoscopy (i.e. photodynamic diagnosis) and narrow band imaging are additional endoscope-based optical imaging modalities that can be combined with CLE to achieve multimodal imaging of the urinary tract. In the future, CLE may be coupled with molecular contrast agents such as fluorescently labeled peptides and antibodies for endoscopic imaging of disease processes with molecular specificity.
View details for DOI 10.3791/4409
View details for PubMedID 23354133
A Cell Phone-Based Microphotometric System for Rapid Antimicrobial Susceptibility Testing.
Journal of laboratory automation
This study demonstrates a low-cost, portable diagnostic system for rapid antimicrobial susceptibility testing in resource-limited settings. To determine the antimicrobial resistance phenotypically, the growth of pathogens in microwell arrays is detected under different antibiotic conditions. The use of a colorimetric cell viability reagent is shown to significantly improve the sensitivity of the assay compared with standard absorbance spectroscopy. Gas-permeable microwell arrays are incorporated for facilitating rapid bacterial growth and eliminating the requirement of bulky supporting equipment. Antibiotics can also be precoated in the microwell array to simplify the assay protocol toward point-of-care applications. Furthermore, a low-cost cell phone-based microphotometric system is developed for detecting the bacterial growth in the microwell array. By optimizing the operating conditions, the system allows antimicrobial susceptibility testing for samples with initial concentrations from 10(1) to 10(6) cfu/mL. Using urinary tract infection as the model system, we demonstrate rapid antimicrobial resistance profiling for uropathogens in both culture media and urine. With its simplicity and cost-effectiveness, the cell phone-based microphotometric system is anticipated to have broad applicability in resource-limited settings toward the management of infectious diseases caused by multidrug-resistant pathogens.
View details for PubMedID 23697894
New Optical Imaging Technologies for Bladder Cancer: Considerations and Perspectives
JOURNAL OF UROLOGY
2012; 188 (2): 361-368
Bladder cancer presents as a spectrum of different diatheses. Accurate assessment for individualized treatment depends on initial diagnostic accuracy. Detection relies on white light cystoscopy accuracy and comprehensiveness. Aside from invasiveness and potential risks, white light cystoscopy shortcomings include difficult flat lesion detection, precise tumor delineation to enable complete resection, inflammation and malignancy differentiation, and grade and stage determination. Each shortcoming depends on surgeon ability and experience with the technology available for visualization and resection. Fluorescence cystoscopy/photodynamic diagnosis, narrow band imaging, confocal laser endomicroscopy and optical coherence tomography address the limitations and have in vivo feasibility. They detect suspicious lesions (photodynamic diagnosis and narrow band imaging) and further characterize lesions (optical coherence tomography and confocal laser endomicroscopy). We analyzed the added value of each technology beyond white light cystoscopy and evaluated their maturity to alter the cancer course.Detailed PubMed® searches were done using the terms "fluorescence cystoscopy," "photodynamic diagnosis," "narrow band imaging," "optical coherence tomography" and "confocal laser endomicroscopy" with "optical imaging," "bladder cancer" and "urothelial carcinoma." Diagnostic accuracy reports and all prospective studies were selected for analysis. We explored technological principles, preclinical and clinical evidence supporting nonmuscle invasive bladder cancer detection and characterization, and whether improved sensitivity vs specificity translates into improved correlation of diagnostic accuracy with recurrence and progression. Emerging preclinical technologies with potential application were reviewed.Photodynamic diagnosis and narrow band imaging improve nonmuscle invasive bladder cancer detection, including carcinoma in situ. Photodynamic diagnosis identifies more papillary lesions than white light cystoscopy, enabling more complete resection and fewer residual tumors. Despite improved treatment current data on photodynamic diagnosis do not support improved high risk diathetic detection and characterization or correlation with disease progression. Prospective recurrence data are lacking on narrow band imaging. Confocal laser endomicroscopy and optical coherence tomography potentially grade and stage lesions but data are lacking on diagnostic accuracy. Several emerging preclinical technologies may enhance the diagnostic capability of endoscopic imaging.New optical imaging technologies may improve bladder cancer detection and characterization, and transurethral resection quality. While data on photodynamic diagnosis are strongest, the clinical effectiveness of these technologies is not proven. Prospective studies are needed, particularly of narrow band imaging, confocal laser endomicroscopy and optical coherence tomography. As each technology matures and new ones emerge, cost-effectiveness analysis must be addressed in the context of the various bladder cancer types.
View details for DOI 10.1016/j.juro.2012.03.127
View details for Web of Science ID 000306270600006
View details for PubMedID 22698620
Rapid hybridization of nucleic acids using isotachophoresis
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2012; 109 (28): 11127-11132
We use isotachophoresis (ITP) to control and increase the rate of nucleic acid hybridization reactions in free solution. We present a new physical model, validation experiments, and demonstrations of this assay. We studied the coupled physicochemical processes of preconcentration, mixing, and chemical reaction kinetics under ITP. Our experimentally validated model enables a closed form solution for ITP-aided reaction kinetics, and reveals a new characteristic time scale which correctly predicts order 10,000-fold speed-up of chemical reaction rate for order 100 pM reactants, and greater enhancement at lower concentrations. At 500 pM concentration, we measured a reaction time which is 14,000-fold lower than that predicted for standard second-order hybridization. The model and method are generally applicable to acceleration of reactions involving nucleic acids, and may be applicable to a wide range of reactions involving ionic reactants.
View details for DOI 10.1073/pnas.1205004109
View details for Web of Science ID 000306642100027
View details for PubMedID 22733732
Endoscopic imaging of Cerenkov luminescence
BIOMEDICAL OPTICS EXPRESS
2012; 3 (6): 1215-1225
We demonstrate feasibility of endoscopic imaging of Cerenkov light originated when charged nuclear particles, emitted from radionuclides, travel through a biological tissue of living subjects at superluminal velocity. The endoscopy imaging system consists of conventional optical fiber bundle/ clinical endoscopes, an optical imaging lens system, and a sensitive low-noise charge coupled device (CCD) camera. Our systematic studies using phantom samples show that Cerenkov light from as low as 1 µCi of radioactivity emitted from (18)F-Fluorodeoxyglucose (FDG) can be coupled and transmitted through conventional optical fibers and endoscopes. In vivo imaging experiments with tumor bearing mice, intravenously administered with (18)F-FDG, further demonstrated that Cerenkov luminescence endoscopy is a promising new tool in the field of endoscopic molecular imaging.
View details for Web of Science ID 000304965700007
View details for PubMedID 22741069
Seeing it through: translational validation of new medical imaging modalities
BIOMEDICAL OPTICS EXPRESS
2012; 3 (4): 764-776
Medical imaging is an invaluable tool for diagnosis, surgical guidance, and assessment of treatment efficacy. The Network for Translational Research (NTR) for Optical Imaging consists of four research groups working to "bridge the gap" between lab discovery and clinical use of fluorescence- and photoacoustic-based imaging devices used with imaging biomarkers. While the groups are using different modalities, all the groups face similar challenges when attempting to validate these systems for FDA approval and, ultimately, clinical use. Validation steps taken, as well as future needs, are described here. The group hopes to provide translational validation guidance for itself, as well as other researchers.
View details for Web of Science ID 000302788200009
View details for PubMedID 22574264
In Situ Electrokinetic Enhancement for Self-Assembled-Monolayer-Based Electrochemical Biosensing
2012; 84 (6): 2702-2707
This study reports a multifunctional electrode approach which directly implements electrokinetic enhancement on a self-assembled-monolayer-based electrochemical sensor for point-of-care diagnostics. Using urinary tract infections as a model system, we demonstrate that electrokinetic enhancement, which involves in situ stirring and heating, can enhance the sensitivity of the strain specific 16S rRNA hybridization assay for 1 order of magnitude and accelerate the time-limiting incubation step with a 6-fold reduction in the incubation time. Since the same electrode platform is used for both electrochemical signal enhancement and electrochemical sensing, the multifunctional electrode approach provides a highly effective strategy toward fully integrated lab-on-a-chip systems for various biomedical applications.
View details for DOI 10.1021/ac203245j
View details for Web of Science ID 000301634500013
View details for PubMedID 22397486
- Next generation of optical diagnostics for bladder cancer using probe-based confocal laser endomicroscopy PHOTONIC THERAPEUTICS AND DIAGNOSTICS VIII, PTS 1 AND 2 2012; 8207
A Universal Electrode Approach for Automated Electrochemical Detection of Bacterial 16S rRNA
6TH IEEE INTERNATIONAL CONFERENCE ON NANO/MOLECULAR MEDICINE AND ENGINEERING (IEEE-NANOMED 2012)
View details for Web of Science ID 000322438500120
Clinical Validation of Integrated Nucleic Acid and Protein Detection on an Electrochemical Biosensor Array for Urinary Tract Infection Diagnosis
2011; 6 (10)
Urinary tract infection (UTI) is a common infection that poses a substantial healthcare burden, yet its definitive diagnosis can be challenging. There is a need for a rapid, sensitive and reliable analytical method that could allow early detection of UTI and reduce unnecessary antibiotics. Pathogen identification along with quantitative detection of lactoferrin, a measure of pyuria, may provide useful information towards the overall diagnosis of UTI. Here, we report an integrated biosensor platform capable of simultaneous pathogen identification and detection of urinary biomarker that could aid the effectiveness of the treatment and clinical management.The integrated pathogen 16S rRNA and host lactoferrin detection using the biosensor array was performed on 113 clinical urine samples collected from patients at risk for complicated UTI. For pathogen detection, the biosensor used sandwich hybridization of capture and detector oligonucleotides to the target analyte, bacterial 16S rRNA. For detection of the protein biomarker, the biosensor used an analogous electrochemical sandwich assay based on capture and detector antibodies. For this assay, a set of oligonucleotide probes optimized for hybridization at 37°C to facilitate integration with the immunoassay was developed. This probe set targeted common uropathogens including E. coli, P. mirabilis, P. aeruginosa and Enterococcus spp. as well as less common uropathogens including Serratia, Providencia, Morganella and Staphylococcus spp. The biosensor assay for pathogen detection had a specificity of 97% and a sensitivity of 89%. A significant correlation was found between LTF concentration measured by the biosensor and WBC and leukocyte esterase (p<0.001 for both).We successfully demonstrate simultaneous detection of nucleic acid and host immune marker on a single biosensor array in clinical samples. This platform can be used for multiplexed detection of nucleic acid and protein as the next generation of urinary tract infection diagnostics.
View details for DOI 10.1371/journal.pone.0026846
View details for Web of Science ID 000296916000027
View details for PubMedID 22066011
Molecular Detection of Bacterial Pathogens Using Microparticle Enhanced Double-Stranded DNA Probes
2011; 83 (16): 6349-6354
Rapid, specific, and sensitive detection of bacterial pathogens is essential toward clinical management of infectious diseases. Traditional approaches for pathogen detection, however, often require time-intensive bacterial culture and amplification procedures. Herein, a microparticle enhanced double-stranded DNA probe is demonstrated for rapid species-specific detection of bacterial 16S rRNA. In this molecular assay, the binding of the target sequence to the fluorophore conjugated probe thermodynamically displaces the quencher probe and allows the fluorophore to fluoresce. By incorporation of streptavidin-coated microparticles to localize the biotinylated probes, the sensitivity of the assay can be improved by 3 orders of magnitude. The limit of detection of the assay is as few as eight bacteria without target amplification and is highly specific against other common pathogens. Its applicability toward clinical diagnostics is demonstrated by directly identifying bacterial pathogens in urine samples from patients with urinary tract infections.
View details for DOI 10.1021/ac2012575
View details for Web of Science ID 000293758800034
View details for PubMedID 21718053
Dynamic Real-time Microscopy of the Urinary Tract Using Confocal Laser Endomicroscopy
2011; 78 (1): 225-231
To develop the diagnostic criteria for benign and neoplastic conditions of the urinary tract using probe-based confocal laser endomicroscopy (pCLE), a new technology for dynamic, in vivo imaging with micron-scale resolution. The suggested diagnostic criteria will formulate a guide for pCLE image interpretation in urology.Patients scheduled for transurethral resection of bladder tumor (TURBT) or nephrectomy were recruited. After white-light cystoscopy (WLC), fluorescein was administered as contrast. Different areas of the urinary tract were imaged with pCLE via direct contact between the confocal probe and the area of interest. Confocal images were subsequently compared with standard hematoxylin and eosin analysis.pCLE images were collected from 66 participants, including 2 patients who underwent nephrectomy. We identified key features associated with different anatomic landmarks of the urinary tract, including the kidney, ureter, bladder, prostate, and urethra. In vivo pCLE of the bladder demonstrated distinct differences between normal mucosa and neoplastic tissue. Using mosaicing, a post hoc image-processing algorithm, individual image frames were juxtaposed to form wide-angle views to better evaluate tissue microarchitecture.In contrast to standard pathologic analysis of fixed tissue with hematoxylin and eosin, pCLE provides real time microscopy of the urinary tract to enable dynamic interrogation of benign and neoplastic tissues in vivo. The diagnostic criteria developed in this study will facilitate adaptation of pCLE for use in conjunction with WLC to expedite diagnosis of urinary tract pathology, particularly bladder cancer.
View details for DOI 10.1016/j.urology.2011.02.057
View details for Web of Science ID 000292080300062
View details for PubMedID 21601243
Comparison of 2.6-and 1.4-mm Imaging Probes for Confocal Laser Endomicroscopy of the Urinary Tract
JOURNAL OF ENDOUROLOGY
2011; 25 (6): 917-921
Probe-based confocal laser endomicroscopy (pCLE) is an emerging technology for dynamic, in vivo imaging of the urinary tract with micron-scale resolution. We conducted a comparative analysis of pCLE with a 2.6-mm probe and a 1.4-mm probe that is compatible with flexible endoscopes.Sixty-seven patients scheduled for bladder tumor resection were recruited. pCLE imaging was performed using 2.6- and 1.4-mm probes. Image quality with the different probes was examined and further compared with standard histopathology.Images with the 2.6-mm probe have better resolution of cell morphology. The 1.4-mm probe has a wider field of view and better view of microarchitecture. While image quality with the 2.6-mm probe is superior, the 1.4-mm probe is compatible with flexible cystoscopy and maneuverability is maintained, enabling imaging of areas of the bladder that were previously challenging to access with the larger probe.The optical specifications of the 2.6-mm probe are more suitable for distinguishing urinary tract histopathology. Further design optimization to improve resolution and additional validation of the diagnostic accuracy of the smaller probe are needed to help extend application of pCLE for optical biopsy of the upper and lower urinary tract.
View details for DOI 10.1089/end.2010.0686
View details for Web of Science ID 000291553500004
View details for PubMedID 21568756
Rapid Detection of Urinary Tract Infections Using Isotachophoresis and Molecular Beacons
2011; 83 (11): 4110-4117
We present a novel assay for rapid detection and identification of bacterial urinary tract infections using isotachophoresis (ITP) and molecular beacons. We applied on-chip ITP to extract and focus 16S rRNA directly from bacterial lysate and used molecular beacons to achieve detection of bacteria specific sequences. We demonstrated detection of E. coli in bacteria cultures as well as in patient urine samples in the clinically relevant range 1E6-1E8 cfu/mL. For bacterial cultures we further demonstrate quantification in this range. The assay requires minimal sample preparation (a single centrifugation and dilution), and can be completed, from beginning of lysing to detection, in under 15 min. We believe that the principles presented here can be used for design of other rapid diagnostics or detection methods for pathogenic diseases.
View details for DOI 10.1021/ac200253x
View details for Web of Science ID 000290978500022
View details for PubMedID 21545089
Biosensor diagnosis of urinary tract infections: a path to better treatment?
TRENDS IN PHARMACOLOGICAL SCIENCES
2011; 32 (6): 330-336
Urinary tract infection (UTI) is among the most common bacterial infections and poses a significant healthcare burden. The standard culture-based diagnosis of UTI has a typical delay of two to three days. In the absence of definitive microbiological diagnosis at the point of care, physicians frequently initiate empirical broad-spectrum antibiotic treatment, and this has contributed to the emergence of resistant pathogens. Biosensors are emerging as a powerful diagnostic platform for infectious diseases. Paralleling how blood glucose sensors revolutionized the management of diabetes, and how pregnancy tests are now conducted in the home, biosensors are poised to improve UTI diagnosis significantly. Biosensors are amenable to integration with microfluidic technology for point-of-care (POC) applications. This review focuses on promising biosensor technology for UTI diagnosis, including pathogen identification and antimicrobial susceptibility testing, and hurdles to be surpassed in the translation of biosensor technology from bench to bedside.
View details for DOI 10.1016/j.tips.2011.03.001
View details for Web of Science ID 000291843800002
View details for PubMedID 21458868
- Advances in the field of urinary tract infections European Urology Today Congress News 2011; 23: 27
- System Integration - A Major Step toward Lab on a Chip JOURNAL OF BIOLOGICAL ENGINEERING 2011; 5 (1)
Hybrid electrokinetic manipulation in high-conductivity media
LAB ON A CHIP
2011; 11 (10): 1770-1775
This study reports a hybrid electrokinetic technique for label-free manipulation of pathogenic bacteria in biological samples toward medical diagnostic applications. While most electrokinetic techniques only function in low-conductivity buffers, hybrid electrokinetics enables effective operation in high-conductivity samples, such as physiological fluids (?1 S m(-1)). The hybrid electrokinetic technique combines short-range electrophoresis and dielectrophoresis, and long-range AC electrothermal flow to improve its effectiveness. The major technical hurdle of electrode instability for manipulating high conductivity samples is tackled by using a Ti-Au-Ti sandwich electrode and a 3-parallel-electrode configuration is designed for continuous isolation of bacteria. The device operates directly with biological samples including urine and buffy coats. We show that pathogenic bacteria and biowarfare agents can be concentrated for over 3 orders of magnitude using hybrid electrokinetics.
View details for DOI 10.1039/c1lc20054b
View details for Web of Science ID 000289951200009
View details for PubMedID 21487576
- Real Time Diagnosis of Bladder Cancer with Probe-Based Confocal Laser Endomicroscopy IMAGING, MANIPULATION, AND ANALYSIS OF BIOMOLECULES, CELLS, AND TISSUES IX 2011; 7902
A Biosensor Platform for Rapid Antimicrobial Susceptibility Testing Directly From Clinical Samples
JOURNAL OF UROLOGY
2011; 185 (1): 148-153
A significant barrier to efficient antibiotic management of infection is that the standard diagnostic methodologies do not provide results at the point of care. The delays between sample collection and bacterial culture and antibiotic susceptibility reporting have led to empirical use of antibiotics, contributing to the emergence of drug resistant pathogens. As a key step toward the development of a point of care device for determining the antibiotic susceptibility of urinary tract pathogens, we report on a biosensor based antimicrobial susceptibility test.For assay development bacteria were cultured with or without antibiotics, and growth was quantitated by determining viable counts and electrochemical biosensor measurement of bacterial 16S rRNA. To determine antibiotic susceptibility directly from patient samples, urine was cultured on antibiotic plates for 2.5 hours and growth was determined by electrochemical measurement of bacterial 16S rRNA. For assay validation 252 urine samples were collected from patients at the Spinal Cord Injury Service at Veterans Affairs Palo Alto Health Care System. The biosensor based antimicrobial susceptibility test was completed for samples containing gram-negative organisms. Pathogen identification and antibiotic susceptibility results were compared between our assay and standard microbiological analysis.A direct biosensor quantitation of bacterial 16S rRNA can be used to monitor bacterial growth for a biosensor based antimicrobial susceptibility test. Clinical validation of a biosensor based antimicrobial susceptibility test with patient urine samples demonstrated that this test was 94% accurate in 368 pathogen-antibiotic tests compared to standard microbiological analysis.This biosensor based antimicrobial susceptibility test, in concert with our previously described pathogen identification assay, can provide culture and susceptibility information directly from a urine sample within 3.5 hours.
View details for DOI 10.1016/j.juro.2010.09.022
View details for Web of Science ID 000285141900047
View details for PubMedID 21074208
System Integration - A Major Step toward Lab on a Chip.
Journal of biological engineering
2011; 5: 6-?
Microfluidics holds great promise to revolutionize various areas of biological engineering, such as single cell analysis, environmental monitoring, regenerative medicine, and point-of-care diagnostics. Despite the fact that intensive efforts have been devoted into the field in the past decades, microfluidics has not yet been adopted widely. It is increasingly realized that an effective system integration strategy that is low cost and broadly applicable to various biological engineering situations is required to fully realize the potential of microfluidics. In this article, we review several promising system integration approaches for microfluidics and discuss their advantages, limitations, and applications. Future advancements of these microfluidic strategies will lead toward translational lab-on-a-chip systems for a wide spectrum of biological engineering applications.
View details for DOI 10.1186/1754-1611-5-6
View details for PubMedID 21612614
Statistical Metamodeling for Revealing Synergistic Antimicrobial Interactions
2010; 5 (11)
Many bacterial pathogens are becoming drug resistant faster than we can develop new antimicrobials. To address this threat in public health, a metamodel antimicrobial cocktail optimization (MACO) scheme is demonstrated for rapid screening of potent antibiotic cocktails using uropathogenic clinical isolates as model systems. With the MACO scheme, only 18 parallel trials were required to determine a potent antimicrobial cocktail out of hundreds of possible combinations. In particular, trimethoprim and gentamicin were identified to work synergistically for inhibiting the bacterial growth. Sensitivity analysis indicated gentamicin functions as a synergist for trimethoprim, and reduces its minimum inhibitory concentration for 40-fold. Validation study also confirmed that the trimethoprim-gentamicin synergistic cocktail effectively inhibited the growths of multiple strains of uropathogenic clinical isolates. With its effectiveness and simplicity, the MACO scheme possesses the potential to serve as a generic platform for identifying synergistic antimicrobial cocktails toward management of bacterial infection in the future.
View details for DOI 10.1371/journal.pone.0015472
View details for Web of Science ID 000284087800026
View details for PubMedID 21124958
Electrochemical immunosensor detection of urinary lactoferrin in clinical samples for urinary tract infection diagnosis
BIOSENSORS & BIOELECTRONICS
2010; 26 (2): 649-654
Urine is the most abundant and easily accessible of all body fluids and provides an ideal route for non-invasive diagnosis of human diseases, particularly of the urinary tract. Electrochemical biosensors are well suited for urinary diagnostics due to their excellent sensitivity, low-cost, and ability to detect a wide variety of target molecules including nucleic acids and protein biomarkers. We report the development of an electrochemical immunosensor for direct detection of the urinary tract infection (UTI) biomarker lactoferrin from infected clinical samples. An electrochemical biosensor array with alkanethiolate self-assembled monolayer (SAM) was used. Electrochemical impedance spectroscopy was used to characterize the mixed SAM, consisted of 11-mercaptoundecanoic acid and 6-mercapto-1-hexanol. A sandwich amperometric immunoassay was developed for detection of lactoferrin from urine, with a detection limit of 145 pg/ml. We validated lactoferrin as a biomarker of pyuria (presence of white blood cells in urine), an important hallmark of UTI, in 111 patient-derived urine samples. Finally, we demonstrated multiplex detection of urinary pathogens and lactoferrin through simultaneous detection of bacterial nucleic acid (16S rRNA) and host immune response protein (lactoferrin) on a single sensor array. Our results represent first integrated sensor platform capable of quantitative pathogen identification and measurement of host immune response, potentially providing clinical diagnosis that is not only more expeditious but also more informative than the current standard.
View details for DOI 10.1016/j.bios.2010.07.002
View details for Web of Science ID 000283804400056
View details for PubMedID 20667707
DYNAMIC REAL TIME MICROSCOPY OF THE URINARY TRACT: AN IMAGING ATLAS BASED ON CONFOCAL LASER ENDOMICROSCOPY
MARY ANN LIEBERT INC. 2010: A278-A278
View details for Web of Science ID 000283864901225
- Matrix Effects-A Challenge Toward Automation of Molecular Analysis JALA 2010; 15 (3): 233-242
Antimicrobial Susceptibility Testing Using High Surface-to-Volume Ratio Microchannels
2010; 82 (3): 1012-1019
This study reports the use of microfluidics, which intrinsically has a large surface-to-volume ratio, toward rapid antimicrobial susceptibility testing at the point of care. By observing the growth of uropathogenic Escherichia coli in gas permeable polymeric microchannels with different dimensions, we demonstrate that the large surface-to-volume ratio of microfluidic systems facilitates rapid growth of bacteria. For microchannels with 250 microm or less in depth, the effective oxygenation can sustain the growth of E. coli to over 10(9) cfu/mL without external agitation or oxygenation, which eliminates the requirement of bulky instrumentation and facilitates rapid bacterial growth for antimicrobial susceptibility testing at the point of care. The applicability of microfluidic rapid antimicrobial susceptibility testing is demonstrated in culture media and in urine with clinical bacterial isolates that have different antimicrobial resistance profiles. The antimicrobial resistance pattern can be determined as rapidly as 2 h compared to days in standard clinical procedures facilitating diagnostics at the point of care.
View details for DOI 10.1021/ac9022764
View details for Web of Science ID 000273983700037
View details for PubMedID 20055494
Laparoscopic Radical Nephrectomy in a Pelvic Ectopic Kidney: Keys to Success
JSLS-JOURNAL OF THE SOCIETY OF LAPAROENDOSCOPIC SURGEONS
2010; 14 (1): 126-129
Laparoscopic radical nephrectomy of a pelvic kidney for renal cell carcinoma is a procedure with little precedent, but one that offers the advantages of the minimally invasive approach. We present our experience with this unique procedure.A 64-year-old male with a history of end-stage renal disease was diagnosed with a 2.6-cm enhancing mass in a pelvic left kidney with 2 separate sources of blood supply. He was offered either an open radical nephrectomy or a laparoscopic radical nephrectomy and opted for the minimally invasive approach.The procedure was performed successfully without complications and with minimal blood loss. The case was marked both by difficulty in mobilizing the sigmoid colon and the limited working space of the pelvis, which made localization of the numerous hilar vessels challenging.Laparoscopic radical nephrectomy for a pelvic ectopic kidney appears to be safe and efficacious. Success is dependent on familiarity with pelvic anatomy, optimal port placement, and preprocedure knowledge of the often-complicated vascular anatomy of the ectopic kidney. Preoperative imaging to delineate anomalous vascular anatomy is mandatory, and ureteral catheter placement is helpful for intraoperative identification purposes.
View details for DOI 10.4293/108680810X12674612765623
View details for Web of Science ID 000278761200023
View details for PubMedID 20529537
- Electrothermal Fluid Manipulation of High-Conductivity Samples for Laboratory Automation Applications Journal of the Association for Laboratory Automation 2010; 15 (6): 426-432
- Nanomedicine: About This Special Issue Journal of the Association for Laboratory Automation 2010; 15 (2): A10
- Matrix Effects?A Challenge Toward Automation of Molecular Analysis Journal of the Association for Laboratory Automation 2010; 15 (3): 233-242
Multiplex Pathogen Identification for Polymicrobial Urinary Tract Infections Using Biosensor Technology: A Prospective Clinical Study
JOURNAL OF UROLOGY
2009; 182 (6): 2735-2741
Rapid diagnosis of urinary tract infection would have a significant beneficial impact on clinical management, particularly in patients with structural or functional urinary tract abnormalities who are highly susceptible to recurrent polymicrobial infections. We examined the analytical validity of an electrochemical biosensor array for rapid molecular diagnosis of urinary tract infection in a prospective clinical study in patients with neurogenic bladder.The electrochemical biosensor array was functionalized with DNA probes against 16S rRNA of the most common uropathogens. Spinal cord injured patients at a Veterans Affairs hospital were recruited into the study. Urine samples were generally tested on the biosensor within 1 to 2 hours of collection. Biosensor results were compared with those obtained using standard clinical microbiology laboratory methods.We successfully developed a 1-hour biosensor assay for multiplex identification of pathogens. From July 2007 to December 2008 we recruited 116 patients, yielding a total of 109 urine samples suitable for analysis and comparison between biosensor assay and standard urine culture. Of the samples 74% were positive, of which 42% were polymicrobial. We identified 20 organisms, of which Escherichia coli, Pseudomonas aeruginosa and Enterococcus species were the most common. Biosensor assay specificity and positive predictive value were 100%. Pathogen detection sensitivity was 89%, yielding a 76% negative predictive value.To our knowledge we report the first prospective clinical study to successfully identify pathogens within a point of care time frame using an electrochemical biosensor platform. Additional efforts to improve the limit of detection and probe design are needed to further enhance assay sensitivity.
View details for DOI 10.1016/j.juro.2009.08.028
View details for Web of Science ID 000271668600072
View details for PubMedID 19837423
A Microfluidic Cartridge System for Multiplexed Clinical Analysis
2009; 14 (6): 407-412
Cartridge-based microfluidics is a promising technology for clinical diagnostics. By miniaturizing the fluid-handling processes required for genomic and proteomic analyses, reagent and specimen volume is minimized along with the size of the system. We demonstrate an automated microfluidic system capable of performing six multiplexed genomic and proteomic analyses simultaneously, by means of an integrated electrochemical sensor and embedded controls.
View details for DOI 10.1016/j.jala.2009.05.002
View details for Web of Science ID 000285163400011
View details for PubMedID 20161584
Optical Biopsy of Human Bladder Neoplasia With In Vivo Confocal Laser Endomicroscopy
JOURNAL OF UROLOGY
2009; 182 (4): 1299-1305
Confocal laser endomicroscopy is a new endoscopic imaging technology that could complement white light cystoscopy by providing in vivo bladder histopathology. We evaluated confocal laser endomicroscopy by imaging normal, malignant appearing and indeterminate bladder mucosa in a pilot study.Patients scheduled to undergo transurethral resection of bladder tumors were recruited during a 3-month period. After standard cystoscopy fluorescein was administered intravesically and/or intravenously as a contrast dye. A 2.6 mm probe based confocal laser endomicroscope was passed through a 26 Fr resectoscope to image normal and abnormal appearing areas. The images were collected with 488 nm excitation at 8 to 12 frames per second. The endomicroscopic images were compared with standard hematoxylin and eosin analysis of transurethral resection of bladder tumor specimens.Of the 27 recruited patients 8 had no cancer, 9 had low grade tumors, 9 had high grade tumors and 1 had a low grade tumor with a high grade focus. Endomicroscopic images demonstrated clear differences between normal mucosa, and low and high grade tumors. In normal urothelium larger umbrella cells are seen most superficially followed by smaller intermediate cells and the less cellular lamina propria. In contrast, low grade papillary tumors demonstrate densely arranged but normal-shaped small cells extending outward from fibrovascular cores. High grade tumors show markedly irregular architecture and cellular pleomorphism.We report the first study to our knowledge of in vivo confocal laser endomicroscopy in the urinary tract. Marked differences among normal urothelium, low grade tumors and high grade tumors were visualized. Pending further clinical investigation and technological improvement, confocal laser endomicroscopy may become a useful adjunct to conventional cystoscopy.
View details for DOI 10.1016/j.juro.2009.06.039
View details for Web of Science ID 000269764100016
View details for PubMedID 19683270
Decreasing Use of Luteinizing Hormone-Releasing Hormone Agonists in the United States is Independent of Reimbursement Changes: A Medicare and Veterans Health Administration Claims Analysis
JOURNAL OF UROLOGY
2009; 182 (1): 255-260
Luteinizing hormone-releasing hormone agonists are the most common form of androgen deprivation therapy in men with prostate cancer. Limited data exist regarding physician decision-making in prescribing luteinizing hormone-releasing hormone agonists. We present an analysis of luteinizing hormone-releasing hormone agonist use trends based on a time matched comparison of data from Medicare and the Veterans Health Administration, a health care system unaffected by recent changes in Medicare reimbursement implemented by the Medicare Modernization Act in 2004.Medicare claims and payment data were obtained from the Centers for Medicare and Medicaid Services from 2003 to 2007 for luteinizing hormone-releasing hormone agonists and for simple orchiectomy. The Veterans Health Administration Pharmacy Benefits Management database was queried for the annual number of prescriptions for luteinizing hormone-releasing hormone agonists during the same period.After implementation of the Medicare Prescription Drug, Improvement and Modernization Act in 2004 the reimbursement of luteinizing hormone-releasing hormone agonists in the Medicare population decreased by 54.8% and annual claims decreased by 25.1% from 2004 to 2007. During the same period luteinizing hormone-releasing hormone agonist use decreased by 16.8% in the Veterans Health Administration population. There was no compensatory increase in the use of simple orchiectomy for androgen deprivation therapy during the study period.Use of luteinizing hormone-releasing hormone agonists has decreased in the Medicare and Veterans Health Administration populations since 2004 without a compensatory increase in the use of alternative forms of androgen deprivation therapy. The shift in practice patterns is likely due to a decrease in Medicare reimbursement for these drugs, an increase in the use of intermittent therapy and increased recognition of the adverse effects associated with androgen deprivation therapy.
View details for DOI 10.1016/j.juro.2009.02.141
View details for Web of Science ID 000266949800108
View details for PubMedID 19450844
- Active Manipulation of Quantum Dots using AC Electrokinetics JOURNAL OF PHYSICAL CHEMISTRY C 2009; 113 (16): 6561-6565
Fibered Confocal Microscopy of Bladder Tumors: An ex Vivo Study
JOURNAL OF ENDOUROLOGY
2009; 23 (2): 197-201
The inadequacy of white-light cystoscopy to detect flat bladder tumors is well recognized. Great interest exists in developing other imaging technologies to augment or supplant conventional cystoscopy. Fibered confocal microscopy offers the promise of providing in vivo histopathologic information to help distinguish malignant from benign bladder lesions. We report the initial use of this technology to visualize tumors in the human bladder.We performed ex vivo fibered confocal imaging of fresh radical cystectomy specimens using the Mauna Kea Technologies Cellvizio system. The findings were compared with results from standard histopathology.The bladders of four patients were imaged using the fibered confocal microscope. Normal and neoplastic urothelium manifested differences in cellular and vascular density.This study demonstrates the feasibility of using fibered confocal microscopy to detect histologic differences between normal and neoplastic urothelium, and establishes a foundation for the use of fiber-based confocal microscopy in clinical studies.
View details for DOI 10.1089/end.2008.0524
View details for Web of Science ID 000263355500005
View details for PubMedID 19196063
A case of prostatic adenocarcinoma recurrence presenting as ductal carcinoma of the prostate
NATURE CLINICAL PRACTICE UROLOGY
2008; 5 (1): 55-58
A 61-year-old man with a history of recurrent prostate cancer presented with obstructive urinary symptoms. He had been diagnosed with locally invasive adenocarcinoma of the prostate 10 years previously and treated with neoadjuvant hormonal and external beam radiation therapies. Because of the patient's rising PSA level, he had been started on goserelin 6 years after this diagnosis and bicalutamide 6 months before the current presentation. The patient presented to the urology clinic with worsening lower urinary tract symptoms consisting of nocturia, urgency, and weak stream.Physical examination revealed a largely normal digital rectal examination, although there was slight asymmetry. The post-void residual urine volume was approximately 200 ml. Laboratory tests showed no evidence of urinary tract infection, but confirmed a rising PSA level despite low serum testosterone levels. Cystoscopic examination revealed hypervascular, large lateral prostatic lobes obstructing the bladder neck. The bladder was normal.The patient underwent transurethral resection of the prostate. Soon after the resection started, bilateral papillary tumors arising from the stroma of both prostatic lobes were uncovered. Owing to the diffuse nature of the papillary tumors, complete resection was not possible. Pathologic analysis confirmed the presence of ductal carcinoma of the prostate.The patient had an uneventful postoperative course and reported improvement of voiding symptoms. Staging with bone scan and CT of the abdomen and pelvis demonstrated multi-focal bony metastasis. The patient was started on docetaxel-based chemotherapy for hormone refractory recurrence of prostate cancer as ductal carcinoma of the prostate. He remains under close surveillance for clinical response and progression of disease.
View details for DOI 10.1038/ncpuro0994
View details for Web of Science ID 000252111100013
View details for PubMedID 18185514
Use of haemostatic agents and glues during laparoscopic partial nephrectomy: A multi-institutional survey from the United States and Europe of 1347 cases
2007; 52 (3): 798-803
Laparoscopic partial nephrectomy (LPN) is a technically challenging procedure for the management of renal tumours. Major complications of LPN include bleeding and urine leakage. Haemostatic agents (HAs) and/or glues may reduce haemorrhage and urine leakage. We sought to examine the current practice patterns for urologists performing LPN with regard to HA use and its relationship with bleeding and urine leakage.A survey was sent via e-mail to urologists currently performing LPN in centres in the United States and Europe. We queried the indications for HA/glue usage, type of HAs/glues used, and whether concomitant suturing/bolstering was performed. In addition, the total number of LPNs performed, laparoscopic tools used to resect the tumour, tumour size, and tumour position were queried.Surveys suitable for analysis were received from 18 centres (n=1347 cases). HAs and/or glues were used in 1042 (77.4%) cases. Mean tumour size was 2.8cm, with 79% of the tumours being defined as exophytic and 21% deep. The HAs and glues used included gelatin matrix thrombin (FloSeal), fibrin gel (Tisseel), bovine serum albumin (BioGlue), cyanoacrylate glue (Glubran), oxidized regenerated cellulose (Surgicel), or combinations of these. Sixteen centres performed concomitant suturing/bolstering. The overall postoperative bleeding requiring transfusion and urine leakage rates were 2.7% and 1.9%, respectively.The use of HAs and/or glues is routine in most centres performing LPN. The overall haemorrhage and urine leakage rates are low following LPN. More studies are needed to assess the potential role of HAs and/or glues in LPN.
View details for Web of Science ID 000249198400028
View details for PubMedID 17329015
Positive margins in laparoscopic partial nephrectomy in 855 cases: A multi-institutional survey from the United States and Europe
JOURNAL OF UROLOGY
2007; 178 (1): 47-50
Open partial nephrectomy has emerged as the standard of care in the management of renal tumors smaller than 4 cm. While laparoscopic radical nephrectomy has been shown to be comparable to open radical nephrectomy with respect to long-term outcomes, important questions remain unanswered regarding the oncological efficacy of laparoscopic partial nephrectomy. We examined the practice patterns and pathological outcomes following laparoscopic partial nephrectomy.A survey was sent to academic medical centers in the United States and in Europe performing laparoscopic partial nephrectomy. The total number of laparoscopic partial nephrectomies, positive margins, indications for intraoperative frozen biopsy as well as tumor size and position were queried.Surveys suitable for analysis were received from 17 centers with a total of 855 laparoscopic partial nephrectomy cases. Mean tumor size was 2.7 cm (+/-0.6). There were 21 cases with positive margins on final pathology, giving an overall positive margin rate of 2.4%. Intraoperative frozen sections were performed selectively at 10 centers based on clinical suspicion of positive margins on excised tumor. Random biopsies were routinely performed on the resection bed at 5 centers. Frozen sections were never performed at 2 centers. Of the 21 cases with positive margins 14 underwent immediate radical nephrectomy based on the frozen section and 7 were followed expectantly.Early experience with laparoscopic partial nephrectomy in this multicenter study demonstrates oncological efficacy comparable to that of open partial nephrectomy with respect to the incidence of positive margins. The practice of intraoperative frozen sections varied among centers and is not definitive in guiding the optimal surgical treatment.
View details for DOI 10.1016/j.juro.2007.03.045
View details for Web of Science ID 000247197000012
View details for PubMedID 17574057
Association of bowel rest and ketorolac analgesia with short hospital stay after laparoscopic donor nephrectomy
2007; 69 (5): 828-831
Because of the shortage of cadaveric kidneys for allograft transplantation, laparoscopic donor nephrectomy is becoming a more feasible option. Several large published series have reported hospital stays as long as 3.3 days. We report the positive effect of preoperative bowel rest and the use of ketorolac for postoperative analgesia on reducing the hospital stay after laparoscopic donor nephrectomy.From 2000 to 2005, 300 patients underwent laparoscopic donor nephrectomy at our institution by a single surgeon (P.G.S.). All patients underwent a bowel preparation regimen involving a clear liquid diet beginning 2 days before surgery. Furthermore, two bottles of magnesium citrate were taken orally the day before surgery, and all patients fasted after midnight before surgery. Patients self-administered one Fleets enema the evening before surgery. Postoperatively, the patients received ketorolac 30 mg intravenously every 6 hours for a maximum of 48 hours, with additional narcotics if necessary for analgesia.The mean operative time was 180 +/- 55 minutes. Typically, patients were admitted the day of surgery and discharged the next postoperative day. The mean donor hospital stay was 1.1 days (range 1 to 3) with no readmissions. More than 97% of our patients were able to tolerate a clear liquid diet, pass flatus, and ambulate the day after surgery.With implementation of a strict bowel preparation regimen and the use of ketorolac for postoperative analgesia, the donor length of stay was markedly improved from previously published results. We attribute the shorter hospital stay to the quicker return of bowel function and to less postoperative discomfort.
View details for DOI 10.1016/j.urology.2007.01.083
View details for Web of Science ID 000248119400008
View details for PubMedID 17482915
Development of an advanced electrochemical DNA biosensor for bacterial pathogen detection
JOURNAL OF MOLECULAR DIAGNOSTICS
2007; 9 (2): 158-168
Electrochemical sensors have the capacity for rapid and accurate detection of a wide variety of target molecules in biological fluids. We have developed an electrochemical sensor assay involving hybridization of bacterial 16S rRNA to fluorescein-modified detector probes and to biotin-modified capture probes anchored to the sensor surface. Signal is generated by an oxidation-reduction current produced by the action of horseradish peroxidase conjugated to an anti-fluorescein monoclonal Fab. A previous study found that this electrochemical sensor strategy could identify uropathogens in clinical urine specimens. To improve assay sensitivity, we examined the key steps that affect the current amplitude of the electrochemical signal. Efficient lysis and release of 16S rRNA from both gram-negative and -positive bacteria was achieved with an initial treatment with Triton X-100 and lysozyme followed by alkaline lysis, resulting in a 12-fold increase in electrochemical signal compared with alkaline lysis alone. The distance in nucleotides between the target hybridization sites of the detector and capture probes and the location of fluorescein modification on the detector probe contributed to a 23-fold change in signal intensity. These results demonstrate the importance of target-probe and probe-probe interactions in the detection of bacterial 16S rRNA using an electrochemical DNA sensor approach.
View details for DOI 10.2353/jmoldx.2007.060052
View details for Web of Science ID 000245427600005
View details for PubMedID 17384207
Complications of laparoscopic living donor nephrectomy and their management: The UCLA experience
2007; 69 (1): 49-52
Because of the shortage of cadaveric kidneys, laparoscopic living donor nephrectomy (LLDN) has become a more common option for transplant recipients. The complication rate has been reported at 6.4% to 16.5%. We present the initial University of California, Los Angeles experience with the complications and their management during LLDN.From January 2000 to December 2005, a single surgeon performed 300 consecutive LLDNs at our institution. A committee of urologists, nephrologists, and support staff approved each donor before surgery. After LLDN was completed, the patients received 30 mg of ketorolac intravenously every 6 hours until discharge. We reviewed the intraoperative and postoperative complications and their management at our institution.Three patients required open conversion, for an overall conversion rate of 1%. Two of the three conversions were a result of a major vascular complication (0.6%). The first major vascular complication resulted from an endovascular stapler malfunction during transection of an accessory left renal artery. The second vascular complication was a Veress needle injury to the left common iliac artery. Three postoperative major complications (1%) occurred, including 1 case of rhabdomyolysis and 2 cases of chylous ascites. Also, 7 minor postoperative complications (2.3%) occurred. Our overall complication rate was 4%. No patients died, and the mean hospital stay was 1.1 days.Our results have shown that LLDN is a safe procedure associated with low morbidity and a quick recovery. Appropriate patient selection is essential to ensure the safety of this procedure.
View details for DOI 10.1016/j.urology.2006.09.030
View details for Web of Science ID 000244114600016
View details for PubMedID 17270612
A Microfluidic System for Rapid Bacterial Pathogen Detection
2007 7TH IEEE CONFERENCE ON NANOTECHNOLOGY, VOL 1-3
View details for Web of Science ID 000261434900301
Laparoscopic renal surgery for benign disease.
Current urology reports
2007; 8 (1): 12-18
Fifteen years after the first report, laparoscopic nephrectomy has demonstrated proven efficacy and safety comparable with an open approach, with a significant advantage of a faster recovery. Wide dissemination of these surgical techniques and continued improvement in instrumentation has made laparoscopy the preferred approach for treating benign pathologic conditions of the kidney. In this review, the expanding indications of laparoscopic simple nephrectomy and the outcomes of the larger clinical series are examined. We discuss the technical aspects of both transperitoneal and retroperitoneal approaches. Finally, laparoscopic cyst decortication and some of the novel applications of laparoscopic renal surgery are highlighted.
View details for PubMedID 17239312
Incidence of ureteral strictures after laparoscopic donor nephrectomy
JOURNAL OF UROLOGY
2006; 176 (3): 1065-1068
Previous reports of laparoscopic donor nephrectomy have suggested that preservation of the gonadal vein with the specimen is important for preventing ureteral strictures. To test this hypothesis we examined our series of patients for the incidence of ureteral strictures when the gonadal vein was not preserved with the specimen during laparoscopic donor nephrectomy.We reviewed the records of 300 consecutive patients at our institution who underwent laparoscopic donor nephrectomy between 2000 and 2005. Mean donor age was 36.7 years (range 18 to 68) in the 167 female and 133 male donors. Mean recipient age was 38.4 years. Average followup was 2 years. During ureteral dissection the gonadal vein was transected just distal to the renal vein and left in situ. The ureter was dissected and transected at the level of the common iliac vessels. Indwelling ureteral stents were used for all recipient ureteral reimplantations and left in place for 1 month. In the postoperative period transplant recipients were followed biweekly for serum creatinine function during month 1 and monthly thereafter. All patients with increased creatinine (greater than 1.3 mg/dl) or an increasing trend were evaluated with transplant renal ultrasound. Clinically significant ureteral stricture was defined as persistent hydronephrosis resulting in impaired renal function and the need for percutaneous nephrostomy tube placement or ureteroscopic management.After laparoscopic living donor transplantation without gonadal vein preservation we found no incidence of clinically significant ureteral stricture.Gonadal vein preservation with the specimen during laparoscopic donor nephrectomy is not necessary. Preservation of the periureteral blood supply is sufficient to prevent ureteral strictures.
View details for DOI 10.1016/j.juro.2006.04.079
View details for Web of Science ID 000239740800054
View details for PubMedID 16890691
Use of electrochemical DNA biosensors for rapid molecular identification of uropathogens in clinical urine specimens
JOURNAL OF CLINICAL MICROBIOLOGY
2006; 44 (2): 561-570
We describe the first species-specific detection of bacterial pathogens in human clinical fluid samples using a microfabricated electrochemical sensor array. Each of the 16 sensors in the array consisted of three single-layer gold electrodes-working, reference, and auxiliary. Each of the working electrodes contained one representative from a library of capture probes, each specific for a clinically relevant bacterial urinary pathogen. The library included probes for Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Enterocococcus spp., and the Klebsiella-Enterobacter group. A bacterial 16S rRNA target derived from single-step bacterial lysis was hybridized both to the biotin-modified capture probe on the sensor surface and to a second, fluorescein-modified detector probe. Detection of the target-probe hybrids was achieved through binding of a horseradish peroxidase (HRP)-conjugated anti-fluorescein antibody to the detector probe. Amperometric measurement of the catalyzed HRP reaction was obtained at a fixed potential of -200 mV between the working and reference electrodes. Species-specific detection of as few as 2,600 uropathogenic bacteria in culture, inoculated urine, and clinical urine samples was achieved within 45 min from the beginning of sample processing. In a feasibility study of this amperometric detection system using blinded clinical urine specimens, the sensor array had 100% sensitivity for direct detection of gram-negative bacteria without nucleic acid purification or amplification. Identification was demonstrated for 98% of gram-negative bacteria for which species-specific probes were available. When combined with a microfluidics-based sample preparation module, the integrated system could serve as a point-of-care device for rapid diagnosis of urinary tract infections.
View details for DOI 10.1128/JCM.44.2.561-570.2006
View details for Web of Science ID 000235344200042
View details for PubMedID 16455913
- Design of Microfluidic T-Form Mixer Utilizing Pressure Disturbances IEEE Nanotechnology Council Review on Advances of Micro, Nano, and Molecular Systems 2006; 1: 595
A point-of-care micro-laboratory for direct pathogen identification in body fluids
2006 1ST IEEE INTERNATIONAL CONFERENCE ON NANO/MICRO ENGINEERED AND MOLECULAR SYSTEMS, VOLS 1-3
View details for Web of Science ID 000248485802019
Rapid, species-specific detection of uropathogen 16S rDNA and rRNA at ambient temperature by dot-blot hybridization and an electrochemical sensor array
MOLECULAR GENETICS AND METABOLISM
2005; 84 (1): 90-99
Development of rapid molecular approaches for pathogen detection is key to improving treatment of infectious diseases. For this study, the kinetics and temperature-dependence of DNA probe hybridization to uropathogen species-specific sequences were examined. A set of oligonucleotide probes were designed based on variable regions of the 16S gene of the Escherichia coli, Proteus mirabilis, Klebsiella oxytoca, and Pseudomonas aeruginosa. A universal bacterial probe and probes-specific for gram-positive and gram-negative organisms were also included. The oligonucleotide probes discriminated among 16S genes derived from 11 different species of uropathogenic bacteria applied to nylon membranes in a dot-blot format. Significant binding of oligonucleotide probes to target DNA and removal of nonspecific binding by membrane washing could both be achieved rapidly, requiring as little as 10 min. An oligonucleotide probe from the same species-specific region of the E. coli 16S gene was used as a capture probe in a novel electrochemical 16-sensor array based on microfabrication technology. Sequence-specific hybridization of target uropathogen 16S rDNA was detected through horseradish peroxidase acting as an electrochemical transducer via a second, detector probe. The sensor array demonstrated rapid, species-specific hybridization in a time course consistent with the rapid kinetics of the dot-blot hybridization studies. As in the dot-blot hybridization studies, species-specific detection of bacterial nucleic acids using the sensor array approach was demonstrated both at 65 degrees C and at room temperature. These results demonstrate that molecular hybridization approaches can be adapted to rapid, room temperature conditions ideal for an electrochemical sensor array platform.
View details for DOI 10.1016/j.ymgme.2004.11.006
View details for Web of Science ID 000227173700012
View details for PubMedID 15639199
- Microelectromechanical systems in urology UROLOGY 2003; 61 (5): 883-887
Case scenario: 26-year-old male with right scrotal pain and swelling.
Reviews in urology
2002; 4 (1): 43-?
View details for PubMedID 16985652
Pediatric urine testing
PEDIATRIC CLINICS OF NORTH AMERICA
2001; 48 (6): 1425-?
Today, urinalysis is one of the most common clinical tests ordered for adult and pediatric patients. Because urine specimens are usually readily available and are obtained noninvasively, the urine testing is well suited for children. This article discusses the most common urine tests performed in children for screening purposes and also less common tests for diagnosis of specific disorders. Special considerations regarding urine specimen collection in children are discussed. Some simple tests that are underused by clinicians are mentioned, as are some exciting new molecular applications of urine testing.
View details for Web of Science ID 000172285000005
View details for PubMedID 11732123
- Coccidioidomycosis presenting as testicular mass JOURNAL OF UROLOGY 2001; 166 (4): 1396-1396
Sarcomatoid renal cell carcinoma: Biologic behavior, prognosis, and response to combined surgical resection and immunotherapy
JOURNAL OF CLINICAL ONCOLOGY
1999; 17 (2): 523-528
Sarcomatoid variants of renal cell carcinoma (RCC) are aggressive tumors that respond poorly to immunotherapy. We report the outcomes of 31 patients with sarcomatoid RCC treated with a combination of surgical resection and immunotherapy.Patients were identified from the database of the University of California Los Angeles Kidney Cancer Program. We retrospectively reviewed the cases of 31 consecutive patients in whom sarcomatoid RCC was diagnosed between 1990 and 1997. Clinical stage, sites of metastasis, pathologic stage, and type of immunotherapy were abstracted from the medical records. The primary end point analyzed was overall survival, and a multivariate analysis was performed to distinguish any factors conferring an improved survivorship.Twenty-six percent of patients were male and 74% were female, and the median age was 59 years (range, 34 to 73 years). Length of follow-up ranged from 2 to 77 months (mean, 21.4 months). Twenty-eight patients (84%) had known metastases at the time of radical nephrectomy (67% had lung metastases and 40% had bone, 21% had liver, 33% had lymphatic, and 15% had brain metastases). Twenty-five patients (81%) received immunotherapy, including low-dose interleukin (IL)-2-based therapy (five patients), tumor-infiltrating lymphocyte-based therapy plus IL-2 (nine patients), high-dose IL-2-based therapy (nine patients), dendritic cell vaccine-based therapy (one patient), and interferon alpha-based therapy alone (one patient). Two patients (6%) achieved complete responses (median duration, 46+ months) and five patients (15%) achieved partial responses (median duration, 36 months). One- and 2-year overall survival rates were 48% and 37%, respectively. Using a multivariate analysis, age, sex, and percentage of sarcomatoid tumor (< or >50%) did not significantly correlate with survival. Improved survival was found in patients receiving high-dose IL-2 therapy compared with patients treated with surgery alone or any other form of immunotherapy (P = .025). Adjusting for age, sex, and percentage of sarcomatoid tumor, the relative risk of death was 10.4 times higher in patients not receiving high-dose IL-2 therapy. Final pathologic T stage did not correlate significantly with outcome, but node-positive patients had a higher death rate per year of follow-up than did the rest of the population (1.26 v 0.76, Cox regression analysis).Surgical resection and high-dose IL-2-based immunotherapy may play a role in the treatment of sarcomatoid RCCs in select patients.
View details for Web of Science ID 000078384500015
View details for PubMedID 10080595
Pelvi-ureteric junction obstruction treated with Acucise (TM) retrograde endopyelotomy
BRITISH JOURNAL OF UROLOGY
1998; 82 (1): 8-11
To determine the efficacy of retrograde endopyelotomy for the treatment of pelvi-ureteric junction (PUJ) obstruction using the Acucise ureteric balloon cutting catheter.Between February 1995 and July 1997, 13 consecutive patients with primary PUJ obstruction underwent Acucise endopyelotomy at our institution. The mean follow-up was 17.7 months (range 7-33). The success of the procedure was based on objective patency on follow-up diuretic isotopic renography and the subjective resolution of symptoms.The treatment was successful by objective criteria in eight of 13 patients and by subjective criteria in nine. The mean operative duration was 33 min (range 25-45) and all 13 patients were discharged within 24 h of the procedure. There were no major complications, such as vascular injury requiring transfusion. There were no delayed failures, as all failures occurred within 3 months of the procedure. Of the four total failures, two patients have successfully undergone open pyeloplasty and one other was found to have a crossing vessel at the lower pole at the time of the operation.In this small series. Acucise endopyelotomy was a safe procedure that offered effective, expeditious first-line treatment for PUJ obstruction. All failures occurred soon after treatment and did not hinder subsequent open pyeloplasty. Further studies with additional patients and a longer follow-up are warranted to determine the long-term efficacy of this promising new treatment.
View details for Web of Science ID 000074856900003
View details for PubMedID 9698655
- Chromophore assisted laser inactivation of cellular proteins Proc SPIE 1997; 2983: 30-6
Isolation of a cDNA encoding a UV-damaged DNA binding factor defective in xeroderma pigmentosum group E cells
MUTATION RESEARCH-DNA REPAIR
1996; 362 (1): 105-117
XPE binding factor (XPE-BF) is deficient in a subset of patients from xeroderma pigmentosum complementation group E. Binding activity copurifies with a 125 kDa polypeptide (p125) that binds to DNA damaged by ultraviolet (UV) radiation and many other agents. We isolated cDNA encoding a polypeptide with a predicted amino acid sequence that matched the sequences of eleven tryptic peptides derived from digestion of XPE-BF purified from human placenta. In vitro transcription and translation of the cDNA yielded a polypeptide of 125 kDa that bound specifically to UV-damaged DNA. Therefore the cDNA encodes either all or the major component of XPE-BF.
View details for Web of Science ID A1996TP86900012
View details for PubMedID 8538642
CHROMOPHORE-ASSISTED LASER INACTIVATION OF SUBUNITS OF THE T-CELL RECEPTOR IN LIVING CELLS IS SPATIALLY RESTRICTED
PHOTOCHEMISTRY AND PHOTOBIOLOGY
1995; 62 (5): 923-929
Chromophore-assisted laser inactivation (CALI) is a molecular photoablation technique that has been used to elucidate the in vivo roles of specific proteins in neural development. The interpretation of its effects on proteins in living cells relies on knowing how spatially restricted the CALI-induced damage is in vivo. To determine the spatial specificity of CALI in living cells, we have applied CALI to individual subunits of the T-cell receptor (TCR) complex on the surface of 2B4 hybridoma cells in culture and have examined the consequent structural and functional integrity of the TCR-alpha, TCR-beta and CD3-epsilon. The CALI of TCR-beta resulted in the disruption of the beta subunit and also resulted in a small effect on antibody binding alone to the neighboring TCR-alpha but caused no effect on another subunit, CD3-epsilon. Reciprocal experiments directing CALI to TCR-alpha and CD3-epsilon gave consistent results. No effects other than a simple loss of function were observed for any of these CALI experiments. These data demonstrate the extent of CALI-induced damage within a multisubunit complex in living cells and provide greater confidence for the future application of this technique to understanding in vivo function of proteins during complex cellular processes.
View details for Web of Science ID A1995TF81400018
View details for PubMedID 8570733
CHROMOPHORE-ASSISTED LASER INACTIVATION OF PROTEINS IS MEDIATED BY THE PHOTOGENERATION OF FREE-RADICALS
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1994; 91 (7): 2659-2663
Chromophore-assisted laser inactivation (CALI) is a technique that selectively inactivates proteins of interest to elucidate their in vivo functions. This method has application to a wide array of biological questions and an understanding of its mechanism is required for its judicious application. We report here that CALI is not mediated by photoinduced thermal denaturation but by photogenerated free radicals. Thermal diffusion calculations suggest that the temperature changes resulting from CALI are too small to cause thermal denaturation, and Arrhenius plots of CALI are inconsistent with a photothermal mechanism. CALI shows an energy dose reciprocity above a threshold and can be inhibited by free-radical quenchers, thus demonstrating a photochemical mechanism of protein inactivation. The type of quenchers that are effective in inhibiting CALI indicates that the active species is a hydrogen abstractor which is not derived from molecular oxygen. We suggest that the active free-radical species is the hydroxyl radical and its very short lifetime explains the spatial specificity of CALI such that half-maximal damage is effected within 15 A from the dye moiety and no significant damage occurs at 34 A. The data are consistent with free-radical formation resulting from a sequential two-photon process.
View details for Web of Science ID A1994ND60200056
View details for PubMedID 8146171
SPATIAL SPECIFICITY OF CHROMOPHORE ASSISTED LASER INACTIVATION OF PROTEIN FUNCTION
1992; 61 (4): 956-962
Chromophore assisted laser inactivation (CALI) is a new technique that selectively inactivates proteins of interest to elucidate their in vivo functions. This method has application to a wide array of biological questions. An understanding of aspects of the mechanism of CALI is required for its judicious application. A critical concern for CALI is its spatial specificity because nonspecific inactivation of neighboring unbound proteins by CALI is a possibility. We show here that CALI is very dependent on the distance between the chromophore and the protein such that there is no significant effect beyond 60 A. CALI using antibodies can inactivate other proteins through a complex but its efficacy decreases approximately fourfold for each intervening protein. These data imply that CALI is spatially specific and damage to neighboring proteins is unlikely.
View details for Web of Science ID A1992HM25900012
View details for PubMedID 1581504