Iron limitation indirectly reduces the Escherichia coli torCAD operon expression by a reduction of molybdenum cofactor availability.

The expression of most molybdoenzymes in Escherichia coli has so far been revealed to be regulated by anaerobiosis and requires the presence of iron, based on the necessity of the transcription factor FNR to bind one [4Fe-4S] cluster. One exception is trimethylamine-N-oxide reductase encoded by the torCAD operon, which has been described to be expressed independently from FNR. In contrast to other alternative anaerobic respiratory systems, the expression of the torCAD operon was shown not to be completely repressed by the presence of dioxygen. To date, the basis for the O2-dependent expression of the torCAD operon has been related to the abundance of the transcriptional regulator IscR, which represses the transcription of torS and torT, and is more abundant under aerobic conditions than under anaerobic conditions. In this study, we reinvestigated the regulation of the torCAD operon and its dependence on the presence of iron and identified a novel regulation that depends on the presence of the bis-molybdopterin guanine dinucleotide (bis-MGD) molybdenum cofactor . We confirmed that the torCAD operon is directly regulated by the heme-containing protein TorC and is indirectly regulated by ArcA and by the availability of iron via active FNR and Fur, both regulatory proteins that influence the synthesis of the molybdenum cofactor. Furthermore, we identified a novel regulation mode of torCAD expression that is dependent on cellular levels of bis-MGD and is not used by other bis-MGD-containing enzymes like nitrate reductase.IMPORTANCEIn bacteria, molybdoenzymes are crucial for anaerobic respiration using alternative electron acceptors. FNR is a very important transcription factor that represents the master switch for the expression of target genes in response to anaerobiosis. Only Escherichia coli trimethylamine-N-oxide (TMAO) reductase escapes this regulation by FNR. We identified that the expression of TMAO reductase is regulated by the amount of bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor synthesized by the cell itself, representing a novel regulation pathway for the expression of an operon coding for a molybdoenzyme. Furthermore, TMAO reductase gene expression is indirectly regulated by the presence of iron, which is required for the production of the bis-MGD cofactor in the cell.

A comprehensive review on lipid nanocarrier systems for cancer treatment: fabrication, future prospects and clinical trials.

Over the last few decades, cancer has been considered a clinical challenge, being among the leading causes of mortality all over the world. Although many treatment approaches have been developed for cancer, chemotherapy is still the most utilized in the clinical setting. However, the available chemotherapeutics-based treatments have several caveats including their lack of specificity, adverse effects as well as cancer relapse and metastasis which mainly explains the low survival rate of patients. Lipid nanoparticles (LNPs) have been utilized as promising nanocarrier systems for chemotherapeutics to overcome the challenges of the currently applied therapeutic strategies for cancer treatment. Loading chemotherapeutic agent(s) into LNPs improves drug delivery at different aspects including specific targeting of tumours, and enhancing the bioavailability of drugs at the tumour site through selective release of their payload, thus reducing their undesired side effects on healthy cells. This review article delineates an overview of the clinical challenges in many cancer treatments as well as depicts the role of LNPs in achieving optimal therapeutic outcomes. Moreover, the review contains a comprehensive description of the many LNPs categories used as nanocarriers in cancer treatment to date, as well as the potential of LNPs for future applications in other areas of medicine and research.

Core-Shell Nanostructured Drug Delivery Platform Based on Biocompatible Metal-Organic Framework-Ligated Polyethyleneimine for Targeted Hepatocellular Carcinoma Therapy.

Multifunctional nanosized metal-organic frameworks (NMOFs) have advanced rapidly over the past decade to develop drug delivery systems (DDSs). These material systems still lack precise and selective cellular targeting, as well as the fast release of the quantity of drugs that are simply adsorbed within and on the external surface of nanocarriers, which hinders their application in the drug delivery. Herein, we designed a biocompatible Zr-based NMOF with an engineered core and the hepatic tumor-targeting ligand, glycyrrhetinic acid grafted to polyethyleneimine (PEI) as the shell. The improved core-shell serves as a superior nanoplatform for efficient controlled and active delivery of the anticancer drug doxorubicin (DOX) against hepatic cancer cells (HepG2 cells). In addition to their high loading capacity of 23%, the developed nanostructure DOX@NMOF-PEI-GA showed an acidic pH-stimulated response and extended the drug release time to 9 days as well as enhanced the selectivity toward the tumor cells. Interestingly, the DOX-free nanostructures showed a minimal toxic effect on both normal human skin fibroblast (HSF) and hepatic cancer cell line (HepG2), but the DOX-loaded nanostructures exhibited a superior killing effect toward the hepatic tumor, thus opening the way for the active drug delivery and achieving efficient cancer therapy applications.

How an ACE2 mimicking epitope-MIP nanofilm recognizes template-related peptides and the receptor binding domain of SARS-CoV-2.

Here we aim to gain a mechanistic understanding of the formation of epitope-imprinted polymer nanofilms using a non-terminal peptide sequence, i.e. the peptide GFNCYFP (G485 to P491) of the SARS-CoV-2 receptor binding domain (RBD). This epitope is chemisorbed on the gold surface through the central cysteine 488 followed by the electrosynthesis of a ∼5 nm thick polyscopoletin film around the surface confined templates. The interaction of peptides and the parent RBD and spike protein with the imprinted polyscopoletin nanofilm was followed by electrochemical redox marker gating, surface enhanced infrared absorption spectroscopy and conductive AFM. Because the use of non-terminal epitopes is especially intricate, here we characterize the binding pockets through their interaction with 5 peptides rationally derived from the template sequence, i.e. implementing central single amino acid mismatch as well as elongations and truncations at its C- and N- termini. Already a single amino acid mismatch, i.e. the central Cys488 substituted by a serine, results in ca. 15-fold lower affinity. Further truncation of the peptides to tetrapeptide (EGFN) and hexapeptide (YFPLQS) results also in a significantly lower affinity. We concluded that the affinity towards the different peptides is mainly determined by the four amino acid motif CYFP present in the sequence of the template peptide. A higher affinity than that for the peptides is found for the parent proteins RBD and spike protein, which seems to be due to out of cavity effects caused by their larger footprint on the nanofilm surface.

Peptide epitope-imprinted polymer microarrays for selective protein recognition. Application for SARS-CoV-2 RBD protein.

We introduce a practically generic approach for the generation of epitope-imprinted polymer-based microarrays for protein recognition on surface plasmon resonance imaging (SPRi) chips. The SPRi platform allows the subsequent rapid screening of target binding kinetics in a multiplexed and label-free manner. The versatility of such microarrays, both as synthetic and screening platform, is demonstrated through developing highly affine molecularly imprinted polymers (MIPs) for the recognition of the receptor binding domain (RBD) of SARS-CoV-2 spike protein. A characteristic nonapeptide GFNCYFPLQ from the RBD and other control peptides were microspotted onto gold SPRi chips followed by the electrosynthesis of a polyscopoletin nanofilm to generate in one step MIP arrays. A single chip screening of essential synthesis parameters, including the surface density of the template peptide and its sequence led to MIPs with dissociation constants (K D) in the lower nanomolar range for RBD, which exceeds the affinity of RBD for its natural target, angiotensin-convertase 2 enzyme. Remarkably, the same MIPs bound SARS-CoV-2 virus like particles with even higher affinity along with excellent discrimination of influenza A (H3N2) virus. While MIPs prepared with a truncated heptapeptide template GFNCYFP showed only a slightly decreased affinity for RBD, a single mismatch in the amino acid sequence of the template, i.e. the substitution of the central cysteine with a serine, fully suppressed the RBD binding.

Tear proteome analysis in ocular surface diseases using label-free LC-MS/MS and multiplexed-microarray biomarker validation.

We analyzed the tear film proteome of patients with dry eye (DE), meibomian gland dysfunction (MGD), and normal volunteers (CT). Tear samples were collected from 70 individuals. Of these, 37 samples were analyzed using spectral-counting-based LC-MS/MS label-free quantitation, and 33 samples were evaluated in the validation of candidate biomarkers employing customized antibody microarray assays. Comparative analysis of tear protein profiles revealed differences in the expression levels of 26 proteins, including protein S100A6, annexin A1, cystatin-S, thioredoxin, phospholipase A2, antileukoproteinase, and lactoperoxidase. Antibody microarray validation of CST4, S100A6, and MMP9 confirmed the accuracy of previously reported ELISA assays, with an area under ROC curve (AUC) of 87.5%. Clinical endpoint analysis showed a good correlation between biomarker concentrations and clinical parameters. In conclusion, different sets of proteins differentiate between the groups. Apolipoprotein D, S100A6, S100A8, and ceruloplasmin discriminate best between the DE and CT groups. The differences between antileukoproteinase, phospholipase A2, and lactoperoxidase levels allow the distinction between MGD and DE, and the changes in the levels of annexin A1, clusterin, and alpha-1-acid glycoprotein 1, between MGD and CT groups. The functional network analysis revealed the main biological processes that should be examined to identify new candidate biomarkers and therapeutic targets.

A new one-step antigen heterologous homogeneous fluorescence immunoassay for progesterone detection in serum.

A new homogeneous immunoassay for the detection of progesterone was developed to measure its concentration in human serum. We utilized the weak cross-reactivity of a monoclonal anti-progesterone antibody to an analog molecule (in this case ß-estradiol) to create a mixture, in which the fluorescence-labeled antibody (AbF) and quencher-labeled BSA-estradiol (eBSAq) were at optimized equilibrium. At this stage, most antibodies were bound to eBSAq and the fluorescence of AbF was quenched. After adding samples containing free progesterone to the system, these would replace the eBSAq at the antigen-binding site. The fluorescence would be released. In contrast to conventional competitive immunoassays, the fluorescence signal increases with increasing progesterone concentration, greatly simplifying detection and calibration. The performance of the assay was very simple; there was only one mixing step; and other hormones like testosterone, estradiol or dehydroepiandrosterone (DHEA) do not interfere the assay. A wide linear range from 0.1 µg/L to 100 µg/L was achieved in buffer, with a LOD of 0.1 µg/L. In human serum the LOD was 5 µg/L, and the linear range was 5-500 µg/L. For this assay it is important to find the right combination of antibody and cross-reactive antigen. If such a combination could be defined, it is conceivable to apply this assay to a wide range of analytes.

Development of peptidyl lysine dendrons: 1,3-dipolar cycloaddition for peptide coupling and antibody recognition.

A straightforward synthesis strategy to multimerize a peptide mimotopes for antibody B13-DE1 recognition is described based on lysine dendrons as multivalent scaffolds. Lysine dendrons that possess N-terminal alkyne residues at the periphery were quantitative functionalized with azido peptides using click chemistry. The solid-phase peptide synthesis (SPPS) allows preparing the peptide dendron in high purity and establishing the possibility of automation. The presented peptide dendron is a promising candidate as multivalent ligand and was used for antibody B13-DE1 recognition. The binding affinity increases with higher dendron generation without loss of specificity. The analysis of biospecific interaction between the synthesized peptide dendron and the antibody was done via surface plasmon resonance (SPR) technique. The presented results show a promising tool for investigations of antigen-antibody reactions.

Characterization of a new maleimido functionalization of gold for surface plasmon resonance spectroscopy.

Para-maleimidophenyl (p-MP) modified gold surfaces have been prepared by one-step electrochemical deposition and used in surface plasmon resonance (SPR) studies. Therefore, a FITC mimotope peptide (MP1, 12 aa), a human mucin 1 epitope peptide (MUC, 9 aa) and a protein with their specific antibodies were used as model systems. The peptides were modified with an N-terminal cysteine for covalent and directed coupling to the maleimido functionalized surface by means of Michael addition. The coupling yield of the peptide, the binding characteristics of antibody and the unspecific adsorption of the analytes were investigated. The results expand the spectrum of biosensors usable with p-MP by widely used SPR and support its potential to be versatile for several electrochemical and optical biosensors. This allows the combination of an electrochemical and optical read-out for a broad variety of biomolecular interactions on the same chip.

Rational design of artificial ß-strand-forming antimicrobial peptides with biocompatible properties.

Because the intensive use of antibiotics has led to a large variety of resistant bacterial strains, therapeutic measures have become increasingly challenging. In order to ensure reliable treatment of diseases, alternative antimicrobial agents need to be explored. In this context, antimicrobial peptides have been discussed as novel bioactive molecules, which, however, may be limited in their applicability due to their high manufacturing costs and poor pharmacokinetic properties. Consequently, the design of artificial antimicrobial peptides featuring two flanking cationic regions and a hydrophobic center is presented. These sequences led to distinct antimicrobial activity on the same order of magnitude as that of naturally occurring reference peptides but with less cytotoxic or cytostatic drawbacks. Furthermore, a deletion and substitution library revealed the minimal sequence requirements. By analysis of the computed 3D structures of these peptides, a single characteristic ß-strand was identified. This structural motif was pivotal for antimicrobial activity. Consequently, an optimized peptide sequence with antimicrobial and biocompatible properties was derived, and its application was demonstrated in a mixed culture experiment. Thus, it was shown that the optimized artificial antimicrobial peptide is suitable as a therapeutic agent and may be used as template for the development of new antimicrobial peptides with unique secondary structures.

Identification of antimicrobial peptides and immobilization strategy suitable for a covalent surface coating with biocompatible properties.

Bacterial accumulation on solid material displays a major source of biomaterial associated infections, cross contamination, and spreading. To overcome these problems, different investigations on surface modifications for the containment of bacterial adhesion have been done. The aim of this research is the development of a rapid and efficient screening procedure to identify and investigate biologically active peptides in an immobilized state in order to produce an antimicrobial surface coating. We figured out that the antimicrobial mode of action is the most important parameter because only peptides with pronounced membrane disruption abilities displayed meaningful activity in an immobilized state. In addition, we highlighted the influence of the coupling reaction chemistry on the activity and amount of the immobilized peptide. Thereupon we developed an optimized antimicrobial surface coating with unrestricted antimicrobial properties by adjusting the immobilization strategy in combination with lowering the necessary peptide amount. Moreover we demonstrated that this antimicrobial surface coating displayed no cytotoxic activity against a eukaryotic cell line and thereby indicates a promising biocompatibility. Furthermore, different antimicrobial peptides obtained either by chemical peptide synthesis or by recombinant DNA technology were used in this study and their activities as well as their potential applications were discussed.

Self-assembled peptide amphiphiles function as multivalent binder with increased hemagglutinin affinity.

BACKGROUND: A promising way in diagnostic and therapeutic applications is the development of peptide amphiphiles (PAs). Peptides with a palmitic acid alkylchain were designed and characterized to study the effect of the structure modifications on self-assembling capabilities and the multiple binding capacity to hemagglutinin (HA), the surface protein of influenza virus type A. The peptide amphiphiles consists of a hydrophilic headgroup with a biological functionality of the peptide sequence and a chemically conjugated hydrophobic tail. In solution they self-assemble easily to micelles with a hydrophobic core surrounded by a closely packed peptide-shell. RESULTS: In this study the effect of a multiple peptide binding partner to the receptor binding site of HA could be determined with surface plasmon resonance measurements. The applied modification of the peptides causes signal amplification in relationship to the unmodified peptide wherein the high constant specificity persists. The molecular assembly of the peptides was characterized by the determination of critical micelle concentration (CMC) with concentration of 10⁻5 M and the colloidal size distribution. CONCLUSION: The modification of the physico-chemical parameters by producing peptide amphiphiles form monomeric structures which enhances the binding affinity and allows a better examination of the interaction with the virus surface protein hemagglutinin.

Highly-integrated lab-on-chip system for point-of-care multiparameter analysis.

A novel innovative approach towards a marketable lab-on-chip system for point-of-care in vitro diagnostics is reported. In a consortium of seven Fraunhofer Institutes a lab-on-chip system called "Fraunhofer ivD-platform" has been established which opens up the possibility for an on-site analysis at low costs. The system features a high degree of modularity and integration. Modularity allows the adaption of common and established assay types of various formats. Integration lets the system move from the laboratory to the point-of-need. By making use of the microarray format the lab-on-chip system also addresses new trends in biomedicine. Research topics such as personalized medicine or companion diagnostics show that multiparameter analyses are an added value for diagnostics, therapy as well as therapy control. These goals are addressed with a low-cost and self-contained cartridge, since reagents, microfluidic actuators and various sensors are integrated within the cartridge. In combination with a fully automated instrumentation (read-out and processing unit) a diagnostic assay can be performed in about 15 min. Via a user-friendly interface the read-out unit itself performs the assay protocol, data acquisition and data analysis. So far, example assays for nucleic acids (detection of different pathogens) and protein markers (such as CRP and PSA) have been established using an electrochemical read-out based on redoxcycling or an optical read-out based on total internal reflectance fluorescence (TIRF). It could be shown that the assay performance within the cartridge is similar to that found for the same assay in a microtiter plate. Furthermore, recent developments are the integration of sample preparation and polymerase chain reaction (PCR) on-chip. Hence, the instrument is capable of providing heating-and-cooling cycles necessary for DNA-amplification. In addition to scientific aspects also the production of such a lab-on-chip system was part of the development since this heavily affects the success of a later market launch. In summary, the Fraunhofer ivD-platform covers the whole value chain ranging from microfluidics, material and polymer sciences, assay and sensor development to the production and assembly design. In this consortium the gap between diagnostic needs and available technologies can be closed.

Reverse transcription-polymerase chain reaction on a microarray: the integrating concept of "active arrays".

In this report we describe the proof of principle of a reverse transcription polymerase chain reaction (RT-PCR) but on-chip, with immobilized specific primers using a transcriptome of mouse-muscle fibroblasts for detection of muscle-specific expression products of these cells. The isolated total mRNA was directly incubated on an array of immobilized and solubilized specific primers, which allow the amplification of certain muscle-specific RNAs via its immobilized cDNAs. In contrast to others, the immobilized cDNA-products were directly synthesized on the chip by applying covalently bound specific primers. The products were detected by the incorporated and fluorophore-modified specific primers of the subsequently synthezised second strand. In addition, this second-strand served as a further template (like the basically used mRNA) in the subsequent solid-phase-PCR to amplify first-strand cDNA copies at the remaining immobilized specific primer-probes. This is the intrinsic factor of the amplification of certain signals of this application. The specific cDNA templates of genes coding for subunits of the mouse muscle acetylcholine receptor (Chrna1, Chrnb1, Chrnd) and the genes coding for myogenin (Myog), muscle creatine kinase (Ckmm), and ATPase (Atp2a2) were amplified on a biochip by RT-PCR directly from freshly isolated mRNA. The resulting procedure allows the detection of mRNA sequences from less than 5 pg of total RNA preparations.

Functional peptide microarrays for specific and sensitive antibody diagnostics.

Peptide microarrays displaying biologically active small synthetic peptides in a high-density format provide an attractive technology to probe complex samples for the presence and/or function of protein analytes. We present a new approach for manufacturing functional peptide microarrays for molecular immune diagnostics. Our method relies on the efficiency of site-specific solution-phase coupling of biotinylated synthetic peptides to NeutrAvidin (NA) and localized microdispensing of peptide-NA-complexes onto activated glass surfaces. Antibodies are captured in a sandwich manner between surface immobilized peptide probes and fluorescence-labeled secondary antibodies. Our work includes a total of 54 peptides derived from immunodominant linear epitopes of the T7 phage capsid protein, Herpes simplex virus glycoprotein D, c-myc protein, and three domains of the Human coronavirus polymerase polyprotein and their cognate mAbs. By using spacer molecules of different type and length for NA-mediated peptide presentation, we show that the incorporation of a minimum spacer length is imperative for antibody binding, whereas the peptide immobilization direction has only secondary importance for antibody affinity and binding. We further demonstrate that the peptide array is capable of detecting low-picomolar concentrations of mAbs in buffered solutions and diluted human serum with high specificity.

Peptide microarrays with site-specifically immobilized synthetic peptides for antibody diagnostics.

Peptide microarrays bear the potential to discover molecular recognition events on protein level, particularly in the field of molecular immunology, in a manner and with an efficiency comparable to the performance of DNA microarrays. We developed a novel peptide microarray platform for the detection of antibodies in liquid samples. The system comprises site-specific solution phase coupling of biotinylated peptides to NeutrAvidin, localized microdispensing of peptide-NeutrAvidin conjugates onto activated glass slides and a fluorescence immuno sandwich assay format for antibody capture and detection. Our work includes synthetic peptides deduced from amino acid sequences of immunodominant linear epitopes, such as the T7 phage capsid protein, Herpes simplex virus glycoprotein D, c-myc protein and three domains of the Human coronavirus 229E polymerase polyprotein. We demonstrate that our method produces peptide arrays with excellent spot morphology which are capable of specific and sensitive detection of monoclonal antibodies from fluid samples.

Directed immobilization of nucleic acids at ultramicroelectrodes using a novel electro-deposited polymer.

A two-step method for the directed immobilization of nucleic acids at ultramicroelectrodes with micron-size dimensions is described. The approach is based on the immobilization of streptavidin at the surface of carbon or noble metal electrodes within a novel electro-deposited polymer, formed by electropolymerization of the natural compound scopoletin (7-hydroxy-6-methoxy-coumarin) at potentials between 0.4 and 0.7 V vs. Ag/AgCl. Biotin-tagged nucleic acids or proteins are immobilized on top of the modified electrodes in a second step. The new method has some advantages compared to classical electropolymerization approaches (e.g. polypyrrole, polyphenol), because the growing polymer is highly hydrophilic, resulting in efficient incorporation of streptavidin and a high biotin binding capacity of 6 pmol cm(-2). The polymer film seems to be non-conductive but shows good swelling properties in aqueous solutions. The feasibility of the method for the electro-directed biochemical modification of individual microelectrodes has been demonstrated by sequential immobilization of two different single strand oligonucleotides onto interdigitated ultramicroelectrodes. The resulting miniature DNA probe was used for single base mutation detection with two synthetic targets (fluorescence-labeled 17-mer oligomers) by evaluating the fluorescence patterns after hybridisation with the immobilised DNA probes. The new method is useful for the production of microelectrode based DNA chips and for the electro-directed immobilisation of biomolecules at microelectrode structures with high spatial resolution and yield.

Detection of progesterone in whole blood samples.

The progesterone concentration in blood samples can be utilised as a marker for the diagnosis of early pregnancy, endocrinopathy and virilism. Here, we describe a method for progesterone detection and measurement in whole blood samples by a surface sensitive biosensor used in conjunction with an integrated optical grating coupler. This device determines refractive index changes near the biosensor's surface. Hence, biological species bound to a surface layer can be measured in real-time without any label. For the measurements, we have modified the indirect competitive immunoassay principle. The concentration of the progesterone antibody was kept at 1 microg/ml. Progesterone concentration was determined in buffer solution and whole blood in a range between 0.005 and 10 ng/ml. The detection limit was determined to be 3 pM. The relative standard deviation was calculated to be 3.5%.

Real-time determination of telomerase activity in cell extracts using an optical biosensor.

A biosensoric approach has been developed to determine the activity of telomerase in tumor cell lysates. An optical sensor, the grating coupler, was used to monitor the association and dissociation of unlabeled compounds on the sensor surface in real time, by virtue of an evanescent field. An oligonucleotide was immobilized on the surface of the optical biosensor and linked with two other oligonucleotides by complementary sequences in an overlapping manner. The 3'-end of the last one carried the sequence of the telomeric substrate (TS) primer used for elongation by telomerase in the telomeric repeat amplification protocol (TRAP) assay. This primer sequence was phosphorothioate (PS)-modified, which is known to strongly increase the affinity to the primer binding site of telomerase protein and consequently the velocity of the telomerase reaction. We show that the PS primer binds to the modified biosensor and is elongated effectively by the telomerase from HL-60 cell lysates. A synthesis rate of 1 nucleotide/min was determined. The inhibitory effect of peptide nucleic acid (PNA) was shown by using immobilized TS. The velocity of the telomerase reaction was slowed down and the signal intensity was below the signal-to-noise ratio. Most nucleic acid detection systems use amplification steps such as polymerase chain reaction (PCR) to increase the amount of the probe. Since telomerase is a polymerase itself amplification of DNA by PCR is not required. Furthermore, no purification steps were required since all measurements were performed with crude cell extract.

Detection of activity of telomerase in tumor cells using fiber optical biosensors.

Human telomerase plays an important role in the cancerogenesis as it is up-regulated in 80-90% of malignant tumors. Thus, it is considered as a potential cancer marker and relevant target in oncology. Its task is the extension of guanine-rich strands of the telomere using an intrinsic RNA as the template. In this paper we developed a new biosensoric assay based on total internal reflection fluorescence measuring the activity of the telomerase on sensor surface. Two alternatives to determine the telomeric activity are demonstrated without the use of amplifying steps as e.g. PCR. The enzymatic inclusion of FITC-labeled dUTPs should reveal the synthesis process in real-time indicating the elongation of a phosphothioate telomeric substrate (PS/TS)-modified primer. Additionally the elongated strand was detected by hybridization with a FITC-labeled complementary linear DNA probe. As the telomeric guanine-rich single-stranded DNA adopts intramolecular quadruplex structures, it was necessary for the hybridization to linearize the telomeric DNA by increasing the reaction temperature to 48 degrees C. The comparison of the telomerase activity using labeled and unlabeled nucleotides indicated the inhibition effect of the FITC-labeled nucleotides slowing down the synthesis rate of the enzyme. It is shown with the modified biosensor that the PS/TS primer binds the telomerase from the HL-60 cell lysates, effectively elongating the immobilized primer. Furthermore no more purification steps were required as all measurements were performed with crude cell extract.