Magneto-mechanical detection of nucleic acids and telomerase activity in cancer cells

The ultra-sensitive magneto-mechanical detection of DNA, single-base-mismatches in nucleic acids, and the assay of telomerase activity are accomplished by monitoring the magnetically induced deflection of a cantilever functionalized with magnetic beads associated with the biosensing interface. The analyzed M13phi DNA hybridized with the nucleic acid-functionalized magnetic beads is replicated in the presence of dNTPs that include biotin-labeled dUTP. The resulting beads are attached to an avidin-coated cantilever, and the modified cantilever is deflected by an external magnetic field. Similarly, telomerization of nucleic acid-modified magnetic beads in the presence of dNTPs, biotin-labeled dUTP, and telomerase from cancer cell extracts and the subsequent association of the magnetic beads to the cantilever surface results in the lever deflection by an external magnetic field. M13phi DNA is sensed with a sensitivity limit of 7.1 x 10(-20) M by the magneto-mechanical detection method.     

Catalytic beacons for the detection of DNA and telomerase activity

DNA and telomerase activity are detected by a DNAzyme generated upon hybridization and opening of a functional catalytic beacon.     

Label-free detection of telomerase activity in HeLa cells using electrochemical impedance spectroscopy

A simple assay based on electrochemical impedance spectroscopy (EIS) for detection of telomerase activity is developed, and it is demonstrated that the label-free EIS method is capable of detecting the telomerase activity in HeLa cells with a detection limit of 1000 HeLa cells without using any amplification technique.     

Real-time PCR detection of telomerase activity using specific molecular beacon probes

Telomerase is a potentially important biomarker and a prognostic indicator of cancer. Several techniques for assessing telomerase activity, including the telomeric repeat amplification protocol (TRAP) and its modified versions, have been developed. Of these methods, real-time quantitative TRAP (RTQ-TRAP) is considered the most promising. In this work, a novel RTQ-TRAP method is developed in which a telomeric repeats-specific molecular beacon is used. The use of the molecular beacon can improve the specificity of the RTQ-TRAP assay, making the method suitable for studying the overall processivity results and the turnover rate of telomerase. In addition, the real-time, closed-tube protocol used obviates the need for post-amplification procedures, reduces the risk of carryover contamination, and supports high throughput. Its performance in synthetic telomerase products and cell extracts suggests that the developed molecular beacon assay can further enhance the clinical utility of telomerase activity as a biomarker/indicator in cancer diagnosis and prognosis. The method also provides a novel approach to the specific detection of some particular gene sequences to which sequence-specific fluorogenic probes cannot be applied directly. Figure Real-time PCR detection of telomerase activity using specific molecular beacon probes Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Magnetic beads-based electrochemiluminescence assay for rapid and sensitive detection of telomerase activity

Telomerase is a potential cancer marker. We developed a new and robust telomerase activity assay which combines the modified telomere repeat amplification protocol (TRAP) with magnetic beads-based electrochemiluminescence (ECL) detection. The high performance of this assay is related to the determination of telomerase activity from single cell levels, and ECL intensity is linear over the range of 1–1000 HeLa cell equivalents. The proposed telomerase assay offers a highly cost- and time-effective alternative to presently available telomerase assays, which are limited by tedious and complicated post-PCR detection.     

PCR-free electrochemical assay of telomerase activity

In this paper, we report a non-PCR-based electrochemical assay that can detect telomerase activity. Telomerase from HeLa cells may induce telomerization of thiolated primers immobilized on a gold electrode surface. With the telomerization reaction, more and more guanine-rich telomeric repeats are formed, so the electrochemical oxidation signal of guanine at about 1.00 V, which is utilized to indicate the elongated guanine-rich telomeric repeats tethered to the primers, will be increased. This assay method can detect the telomerase activity originated from 3000 HeLa cells and thus holds promise as a simple and sensitive approach in clinical diagnosis of cancer.     

The use of gold nanoparticles in diagnostics and detection

The widespread use of gold nanoparticles (GNPs) as labels in diagnostics and detection is due to a unique combination of chemical and physical properties that allow biological molecules to be detected at low concentrations. In this critical review detection methods based on GNPs are divided up and discussed based on the way in which signals are generated in response to specific target molecules. Particular attention is devoted to methods that allow target molecules to be detected with the unaided eye because these, more than any other, harness the full range of properties that make GNPs unique. Methods that are discussed include those in which specific target molecules induce a visible colour change, chromatographic methods that allow non-specialized users to perform sophisticated tests without additional equipment and methods in which trace amounts of GNPs are rendered visible to the unaided eye by catalytic deposition of a metal such as silver. The use of metal deposition as a means of enhancing the signal for optical and electrical detection is also reviewed. The other detection methods included in this review are based on interactions between GNPs and molecules located in close proximity to their surface. These include methods in which light emission from such molecules is enhanced (surface enhanced Raman scattering) or quenched (fluorescence), and methods in which the accumulation of specific target molecules induce subtle changes in the extinction spectra of GNPs that can be followed in real time with inexpensive equipment (166 references).     

A sensitive fluorescence strategy for telomerase detection in cancer cells based on T7 exonuclease-assisted target recycling amplification

A sensitive fluorescence strategy based on T7 exonuclease-assisted target recycling amplification was developed for telomerase detection in cancer cells. The novel strategy improved the fluorescence signal and sensitivity compared with the previously reported methods.     

Determination of the activity of telomerase in cancer cells by using BSA-protected gold nanoclusters as a fluorescent probe

Gold nanoclusters (AuNCs) protected with a bovine serum albumin (BSA) coating are known to emit red fluorescence (peaking at 650 nm) on photoexcitation with ultraviolet light (365 nm). On addition of Cu(II) ions, fluorescence is quenched because Cu(II) complexes certain amino acid units in the BSA chain. Fluorescence is, however, restored if pyrophosphate (PPi) is added because it will chelate Cu(II) and remove it from the BSA coating on the AuNCs. Because PPi is involved in the function of telomerase, the BSA@AuNCs loaded with Cu(II) can act as a fluorescent probe for determination of the activity of telomerase. A fluorescent assay was worked out for telomerase that is highly sensitive and has a wide linear range (10 nU to 10 fM per mL). The fluorescent probe was applied to the determination of telomerase activity in cervix carcinoma cells via imaging. It is shown that tumor cells can be well distinguished from normal cells by monitoring the differences in intracellular telomerase activity.

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Graphical abstract Gold nanoclusters (AuNCs) protected by bovine serum albumin (BSA) and displaying red photoluminescence were prepared as fluorescent probe for the determination of telomerase activity and used for imaging of cervix carcinoma (HeLa) cells.

     

Evaluation of Raman probe for oesophageal cancer diagnostics

Early detection of (pre-)cancerous changes improves prognosis, therefore in the UK patients at high risk of developing gastrointestinal cancers are enrolled on endoscopic surveillance programmes or the Bowel Cancer Screening Programme. The current gold standard technique for the detection of pre-cancerous changes in the gastrointestinal tract is histopathological analysis of biopsy tissue collected at endoscopy. This relies upon subjective assessment of morphological changes within the excised tissue samples and poor targeting of pre-malignant lesions. Raman spectroscopy offers a number of potential advantages for in vivo assessment of tissue at endoscopy. The performance of a custom built Raman probe as a biopsy targeting tool has been evaluated using excised biopsy material. Multivariate classification models have been used to demonstrate the likely ability of a miniature, confocal, fibre optic Raman probe to be used as an optical biopsy tool at endoscopy to provide spectral information in clinically practicable timescales. This technique could facilitate improved targeting of excisional biopsy with associated clinical benefits.     

Optimized pH-responsive cyanine fluorochromes for detection of acidic environments

Modulation of pH-responsive cyanine dye pK(a) values via heteroatom substitution allows for design of fluorescent reporters that are tuned for potential imaging of biologically relevant acidic environments.     

Assays for human telomerase activity: progress and prospects

Human telomerase is a ribonucleoprotein complex that functions as a telomere terminal transferase by adding multiple TTAGGG hexamer repeats using its integral RNA as the template. There is a very strong association between telomerase activity and malignancy in nearly all types of cancer, suggesting that telomerase could be used not only as a diagnostic and prognostic marker but also as a therapeutic target for managing cancer. The significant progress in biomedical telomerase research has necessitated the development of new bioanalytical methods for the rapid, sensitive, and reliable detection of telomerase activity in a particular cell or clinical tissue and body fluids. In this review, we highlight some of the latest methods for identifying telomerase activity and inhibition and discuss some of the challenges for designing innovative telomerase assays. We also summarise the current technologies and speculate on future directions for telomerase testing.     

Gadolinium-based cancer therapeutic liposomes for chemotherapeutics and diagnostics

Gadolinium (Gd)-based cancer therapeutic liposomes can be used for chemotherapeutics and diagnostics. In this study, dual functional liposomes co-encapsulating doxorubicin (Dox) and Gd were prepared by Dox-transition metal complexation. Preparation conditions were optimized to obtain liposomes containing high concentrations of Dox and Gd. The optimized liposomes Gd250 co-encapsulated 3.6 mM of Dox and 1.9 mM of Gd. The magnetic resonance (MR) properties of Gd250 liposomes were determined using a 4.7 T MR system. Cellular uptake of Dox was determined using a flow cytometer and a confocal microscopy and that of Gd was measured using an inductively coupled plasma-atomic emission spectrometer. Although encapsulated Gd exhibited lower relaxivity than MRbester?, which is widely used for clinical diagnosis, because of limited diffusion across the liposome membrane, Gd250 liposomes showed much higher cellular uptake than that of MRbester?. In Gd250 liposomes, Gd was highly accumulated in B16F10 cells, which could provide improved contrast sensitivity for molecular imaging. Additionally, in Gd250 liposomes, Dox was highly internalized, which could enhance its cancer therapeutic effects. Consequently, we suggest that dual functional liposomes can be used as therapeutic and diagnostic carriers.     

Mathematical modeling of an NMR chemistry problem in ovarian cancer diagnostics

We use mathematical modeling via the fast Padé transform (FPT) with respect to a theoretically-designed problem based on time signals that are similar to NMR data as encoded from benign and malignant ovarian cyst fluid. The FPT reconstructed exactly all the input spectral parameters by using exceedingly small fractions of the full time signals both for those corresponding to the benign, as well as to the malignant case. The converged parametric results remained stable thereafter at longer signal lengths. The Padé absorption spectra yielded clear resolution of all the extracted physical metabolites. The capacity of the FPT to resolve and precisely quantify the physical resonances as encountered in benign versus malignant ovarian cystic fluid is demonstrated. The practical significance of such findings is enhanced by the avoidance of the time signals’ exponential tail which is embedded in the background, leading to problems in quantification. Without any fitting or numerical integration of peak areas, the FPT reliably yields the metabolite concentrations of major importance for distinguishing benign from malignant ovarian lesions. Thus, the FPT provides distinct advantages relative to the standard Fourier methodology, which is also stable, but has a number of drawbacks. These include limited resolution capacity, as well as non-parametric estimation, so that only a shape spectrum is generated and post-processing is necessary via, e.g., fitting or numerical integrations which are not unique. The FPT is also distinguished from other competitive parametric methods, which are generally unstable as a function of signal length N at a fixed bandwidth and, therefore, particularly unsuitable to clinical data. We conclude that these advantages of the FPT could be of definite benefit for ovarian cancer diagnostics via NMR and that this line of investigation should continue with encoded data from benign and malignant ovarian tissue, in vitro and in vivo. This avenue is of clinical urgency for early ovarian cancer detection, a goal which is still elusive and achievement of which would confer a major survival benefit.     

Multiplexed tyrosine kinase activity detection in cancer cells using a hydrogel immobilized substrate

Kinases play a key role in cellular signaling, and the overactivation or overexpression of these kinases has been linked to a variety of cancers. Tyrosine kinase inhibitors treat the mechanism of these cancers by targeting the specific kinases that are overactive. Some patients, however, do not respond to these inhibitors or develop resistance to these inhibitors during treatment. Additionally, even within cancers of the same tissue type, different kinases may be overactive in different patients. For example, some lung cancers overexpress epidermal growth factor receptor (EGFR) and respond to EGFR inhibitors, whereas other lung cancers do not overexpress EGFR and receive no benefit from this treatment. Even among patients exhibiting EGFR overexpression, some do not respond to EGFR kinase inhibitors because other kinases, such as Met kinase, are also overactivated. Here we describe a quantitative and specific multiplexed microfluidic assay using a hydrogel immobilized substrate for measuring the kinase activity of Met and Abl kinase from cancer cells. We immobilized kinase-specific substrates on macroporous hydrogel micropillars in microchannels. These microchannels were incubated with 6 μl of a kinase reaction solution containing cancer cell lysate, and we measured kinase activity via fluorescence detection of a phosphotyrosine antibody. We showed that the assay can specifically measure the activity of both Met and Abl kinase within one microchannel and has the potential to measure the activity of as many as five kinases within one microchannel. The assay also detected Met kinase inhibition from lysates of cancer cells grown in the Met kinase inhibitor PHA665752.

Role of direct bioautographic method for detection of antistaphylococcal activity of essential oils

The aim of the present study was the chemical characterization of some traditionally used and therapeutically relevant essential oils (thyme, eucalyptus, cinnamon bark, clove, and tea tree) and the optimized microbiological investigation of the effect of these oils on clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA). The chemical composition of the oils was analyzed by TLC, and controlled by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The antibacterial effect was investigated using a TLC-bioautographic method. Antibacterial activity of thyme, clove and cinnamon oils, as well as their main components (thymol, carvacrol, eugenol, and cinnamic aldehyde) was observed against all the bacterial strains used in this study. The essential oils of eucalyptus and tea tree showed weak activity in the bioautographic system. On the whole, the antibacterial activity of the essential oils could be related to their most abundant components, but the effect of the minor components should also be taken into consideration. Direct bioautography is more cost-effective and better in comparison with traditional microbiological laboratory methods (e.g. disc-diffusion, agar-plate technique).     

Facile assay of telomerase activity utilizing a DNA-detectable chemiluminogenic reagent.

Telomerase shows increased activity in most human cancers and germ line cells, but not in normal human somatic cells. We describe a novel chemiluminescence method for the facile assay of telomerase activity in human cells. The telomerase substrate was incubated with the cell lysate containing various amounts of telomerase, and then the telomerase product was amplified by the polymerase-chained reaction (PCR). The PCR products were separated from the excess substrate, primer and deoxyribonucleotide triphosphates by a centrifugal filter, which distinguished different molecular sizes. The isolated products were reacted with a DNA-detectable chemiluminogenic reagent, 3,4,5-trimethoxyphenylglyoxal. The proposed assay method gave linearity for the telomerase activity in 100 to 10000 cells (r2=0.997), and allowed the assay not only of lower activity, but also of higher activity of telomerase without the requirement of any special labeled-PCR primers in the assay system.