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.

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.

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.

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 E. 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 micrometer 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 hours compared to days in standard clinical procedures facilitating diagnostics at the point of care.

Abstract. A novel gene, designated byr4, was identified in Schizosaccharomyces pombe that affects the mitotic cell cycle and shows genetic interactions with the rasl signaling pathways. Null alleles of byr4 cause cell cycle arrest in late mitosis and permit multiple rounds of septation. The multiple septa typically divide two nuclei, but the nuclei frequently do not stain equally with 4',6-diamidino-2-phenylindole (DAPI), suggesting that byr4 is required for proper karyokinesis. Overexpression of byr4 inhibits cytokinesis, but cell cycle progression continues leading to multinucleate cells. When byr4 is overexpressed, the early steps in the cytokinesis pathway, including formation of the medial F-actin ring, occur normally; however, the later steps in the pathway, including contraction of the F-actin ring, septation, and rearrangement of the medial F-actin following mitosis, rarely occur, byr4 shows two genetic interactions with rasl. The inhibition of cytokinesis by byr4 overexpression was exacerbated by null alleles of rasl and scdl, suggesting a link between pathways needed for cell polarity and cytokinesis. Overexpression of byr4 also partially bypasses the need for rasl for sporulation. The electrophoretic mobility of the byr4 protein varied in response to mutants that perturb cytokinesis and karyokinesis, suggesting interactions between byr4 and these gene products. A more rapidly migrating byr4 protein was found in cells with mutations in cdc16, which undergo repeated septation, and in cdcl5, which fail to form a medial F-actin ring in mitosis. A slower migrating byr4 protein was found in cells with a mutation in the 13-tubulin gene, which arrests cells at the metaphase-anaphase transition. T Hc~ ras proteins are GTPases that cycle between anive, GTP-bound form and an inactive, GDP-.It. bound form (Boguski and McCormick, 1993). Depending on the cellular context, activated ras can stimulate the cell division cycle, alter cell shape, or cause cellular differentiation (Bourne et al., 1990). Several pathways are implicated in signaling downstream of mammalian ras proteins. The best characterized pathway activates the raf kinase. Activated ras recruits the raf kinase and activated raf, in turn, activates a mitogen-activated protein (MAP) 1 kinase cascade (Herskowitz, 1995;Marshall, 1995). A second ras effector may be phosphatidylinositol(PI)-3 kinase. Activated ras binds to PI-3 kinase and ras binding stimulates PI-3 kinase activity four-fold in vitro (RodriguezVinciana et al., 1994). An activated allele of PI-3 kinase, however, activates ras and requires ras for signaling, suggesting ras is an effector of PI-3 kinase (Hu et al., 1995). A third ras effector may be ralGDS, a positive regulator of the ral GTPase (Hofer et al., 1994;Kikuchi et al., 1994;Spaargaren and Bischoff, 1994). Activated ral GTPase Address all correspondence to Charles F. Albright, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, 343-2174; Fax: (615) 343-0704. 1. Abbreviations used in this paper: DAPI,...

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.

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, which has contributed to the emergence of resistant pathogens. Biosensors are emerging as a powerful diagnostic platform for infectious diseases. Similar to how blood glucose sensors revolutionized the management of diabetes and pregnancy tests are now conducted at home, biosensors are poised to significantly improve UTI diagnosis. Biosensors are amenable to integration with microfluidic technology for point-of-care applications. This review focuses on promising biosensor technology for UTI diagnosis, including pathogen identification and antimicrobial susceptibility testing and hurdles in the translation of biosensor technology from bench to bedside.

Purpose 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. Materials and Methods 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. Results 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. Conclusions 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.