Quantitative assessment of in vivo HIV protease activity using genetically engineered QD-based FRET probes.

HIV protease plays a central role in its life cycle leading to release of functional viral particles. It has been successfully used as a therapeutic target to block HIV infection. Several protease inhibitors (PIs) are currently being employed as a part of anti-HIV therapy. However, the constant genetic drift in the virus leads to accumulation of mutations in both cleavage site and the protease, resulting in resistance and failure of therapy. We reported the use of a quantum dot (QD)-based protein probe for the in vivo monitoring of HIV-1 protease activity based on fluorescence resonance energy transfer. In the current study, we demonstrate the utility of this approach by quantifying the in vivo cleavage rates of three known protease and cleavage site mutations in the presence or absence of different PIs. The changes in IC50 values for the different PIs were similar to that observed in patients, validating our assay as a rapid platform for PI screening.

Detection of RNA viruses: current technologies and future perspectives.

RNA viruses constitute one of the major classes of pathogenic organisms causing human diseases, with varying degrees of severity. This review summarizes the conventional and emerging technologies that are available for the detection of these organisms. Cell culture-based techniques for viral detection have been popular since their inception and continue to be the gold standard against which all other techniques. Over many years, these techniques have undergone some radical changes, reducing the total time needed for detection and improving sensitivity, although even with their reliability and improved features they are being slowly replaced by nucleic acid-based technologies. These molecular detection techniques have revolutionized the area of viral detection by their high sensitivity, selectivity, and short detection time. The majority of nucleic acid-based techniques depend on amplifying viral RNA; however, there are some newer emerging techniques that detect viral RNA in live cells using various configurations of florescent probes. In addition, nucleic acid-based technology has made it possible for multiviral detection with either multiplex polymerase chain reaction assays or microarrays. Every technique described in this review has its own unique abilities, making them indispensable for viral detection. However, we believe that nucleic acid-based technologies will find widespread use after being standardized, limiting other technologies to very specific uses.

Detecting RNA viruses in living mammalian cells by fluorescence microscopy.

Traditional methods that rely on viral isolation and culture techniques continue to be the gold standards used for detection of infectious viral particles. However, new techniques that rely on visualization of live cells can shed light on understanding virus-host interaction for early stage detection and potential drug discovery. Live-cell imaging techniques that incorporate fluorescent probes into viral components provide opportunities for understanding mRNA expression, interaction, and virus movement and localization. Other viral replication events inside a host cell can be exploited for non-invasive detection, such as single-virus tracking, which does not inhibit viral infectivity or cellular function. This review highlights some of the recent advances made using these novel approaches for visualization of viral entry and replication in live cells.

A quantum-dot based protein module for in vivo monitoring of protease activity through fluorescence resonance energy transfer.

Here, we present a new generation of nanoscale probes for in vivo monitoring of protease activity by fluorescence resonance energy transfer (FRET). The approach is based on a genetically programmable protein module carrying a fluorescently labeled, protease-specific sequence that can self-assemble onto quantum dots. The protein module was used for real-time detection of human immunodeficiency virus type-1 protease (HIV-1 Pr) activity as well as quantitative assessment of inhibitor efficiency.

Single-walled carbon nanotubes chemiresistor aptasensors for small molecules: picomolar level detection of adenosine triphosphate.

Here we report single walled-carbon nanotubes (SWNTs)-based chemiresistor aptasensors for highly sensitive and selective detection of weakly or uncharged molecules using the displacement format. As a proof-of-concept we demonstrate the detection of ATP, a small weakly charged molecule, by displacement of the ssDNA anti-ATP aptamer hybridized to a small capture oligonucleotide covalently attached on SWNTs, with picomolar sensitivity and selectivity over GTP.

Carbon nanotubes-based chemiresistive biosensors for detection of microorganisms.

The paper reports carbon nanotube (CNT)-based immunosensors for the detection of two types of microorganisms, bacteria and viruses. The pathogen Escherichia coli O157:H7 and the bacteriophage T7 were selected as model for bacteria and viruses, respectively, while E. coli K12 and the bacteriophage MS2 were used to assess the selectivity of the biosensor. The transduction element consisted of single-walled carbon nanotubes aligned in parallel bridging two gold electrodes to function as a chemiresistive biosensor. Single-walled carbon nanotubes (SWNTs) were functionalized with specific antibodies (Ab) for the different microorganisms by covalent immobilization to the non-covalently bound 1-pyrene butanoic acid succinimidyl ester. A significant increase in the resistance of the device was observed when the biosensor was exposed to E. coli O157:H7 whole cells or lysates with a limit of detection of 10(5) and 10(3) CFU (colony forming units)/mL, corresponding to 10(3) and 10(1) CFU/chip, respectively, while no response was observed when the biosensor was exposed to the E. coli K12. In the case of virus detection, a significant resistance increase was detected due to interaction of the bacteriophages with the Abs, with a limit of detection of 10(3) PFU/mL corresponding to 10(1)PFU/chip and excellent selectivity against MS2 bacteriophage. The sensor exhibited a fast response time of ∼5 min in the case of bacteriophage detection, while the response time for the detection of bacteria was 60 min.

Single-walled carbon nanotube chemoresistive label-free immunosensor for salivary stress biomarkers.

The present work is focused on the development, analytical characterization and evaluation of selective and sensitive SWNT-chemiresistor immunosensor for the label-free detection of salivary α-amylase (SAA). SWNTs were aligned to bridge lithographically patterned gold microelectrodes using AC dielectrophoresis followed by functionalization with anti-SAA antibodies. The nano-immunosensors exhibited excellent sensitivity over the clinically relevant range (19 to 308 U ml(-1)) with a limit of detection (LOD) of 6 µg ml(-1) (0.6 U ml(-1)) and 7.8 µg ml(-1) (0.78 U ml(-1)) in phosphate buffer and artificial saliva, respectively, and no interference from other components of saliva. This label-free nano-immunosensor technology has potential application in clinical diagnosis for stress biomarkers expressed in human saliva at the point-of-care.

Carbon nanotubes-based chemiresistive immunosensor for small molecules: detection of nitroaromatic explosives.

In recent years, there has been a growing focus on use of one-dimensional (1-D) nanostructures, such as carbon nanotubes and nanowires, as transducer elements for label-free chemiresistive/field-effect transistor biosensors as they provide label-free and high sensitivity detection. While research to-date has elucidated the power of carbon nanotubes- and other 1-D nanostructure-based field effect transistors immunosensors for large charged macromolecules such as proteins and viruses, their application to small uncharged or charged molecules has not been demonstrated. In this paper we report a single-walled carbon nanotubes (SWNTs)-based chemiresistive immunosensor for label-free, rapid, sensitive and selective detection of 2,4,6-trinitrotoluene (TNT), a small molecule. The newly developed immunosensor employed a displacement mode/format in which SWNTs network forming conduction channel of the sensor was first modified with trinitrophenyl (TNP), an analog of TNT, and then ligated with the anti-TNP single chain antibody. Upon exposure to TNT or its derivatives the bound antibodies were displaced producing a large change, several folds higher than the noise, in the resistance/conductance of SWNTs giving excellent limit of detection, sensitivity and selectivity. The sensor detected between 0.5 ppb and 5000 ppb TNT with good selectivity to other nitroaromatic explosives and demonstrated good accuracy for monitoring TNT in untreated environmental water matrix. We believe this new displacement format can be easily generalized to other one-dimensional nanostructure-based chemiresistive immuno/affinity-sensors for detecting small and/or uncharged molecules of interest in environmental monitoring and health care.

Single-walled carbon nanotube-based chemiresistive affinity biosensors for small molecules: ultrasensitive glucose detection.

We report for the first time single-walled carbon nanotube (SWNT)-based chemiresistive affinity sensors for highly sensitive and selective detection of small and/or weakly charged or uncharged molecules using a displacement format. The detection of glucose, a small, weakly charged molecule, by displacement of plant lectin (concavalin A) bound to a polysaccharide (dextran) immobilized on SWNTs with picomolar sensitivity and selectivity over other sugars and human serum proteins is demonstrated as a proof of concept.

Nano aptasensor for protective antigen toxin of anthrax.

We demonstrate a highly sensitive nano aptasensor for anthrax toxin through the detection of its polypeptide entity, protective antigen (PA toxin) using a PA toxin ssDNA aptamer functionalized single-walled carbon nanotubes (SWNTs) device. The aptamer was developed in-house by capillary electrophoresis systematic evolution of ligands by exponential enrichment (CE-SELEX) and had a dissociation constant (K(d)) of 112 nM. The aptasensor displayed a wide dynamic range spanning up to 800 nM with a detection limit of 1 nM. The sensitivity was 0.11 per nM, and it was reusable six times. The aptasensor was also highly selective for PA toxin with no interference from human and bovine serum albumin, demonstrating it as a potential tool for rapid and point-of-care diagnosis for anthrax.