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Hypoxia plays a crucial role in cancer progression, and it has great significance for monitoring hypoxic level in biosystems. Cancer stem cells (CSCs) represent a small population of tumour cells that regard as the key to seed tumours. The survival of CSCs depend on the tumour microenvironment, which is distinct region has the hypoxic property. Therefore, the detection of the hypoxic CSC niche plays a pivotal role in the destructing the ‘soil’ of CSCs, and eliminating CSCs population. Numerous one-photon excited fluorescent probes have been developed to indicate the hypoxic status in tumours through the detection of nitroreductase (NTR) level. However, the biomedical application of one-photon fluorescent probes is limited due to the poor tissue penetration. In the present work, we reported a two-photon fluorescent probe to detect the NTR in CSCs and monitor the hypoxic microenvironment in vivo. The two-photon fluorescent molecular probe with a hypoxic specific response group can be reduced by NTR under hypoxic conditions. We used the two-photon probe to detect the hypoxia status of 3D cultured-CSCs in vitro and in vivo CSCs' microenvironment in tumour. The two-photon absorption cross section extends fluorescent excitation spectra to the near infrared region, which dramatically promotes the tissue penetration for hypoxic microenvironment detection of CSC in vivo.

As one of the most important post-translational modifications (PTMs) of peptidome, glycopeptidome is closely related to serious disease, especially to cancer. In order to specifically discover and analyze glycopeptidome biomarkers for clinical diagnosis of cancer on early-stage, it is crucial to develop efficient technique to analyze low-abundance of endogenous glycopeptides in biological samples. In this report, a hydrophilic probe in mesoporous pore (denoted as Fe3O4@mSiO2@G6P) was designed and prepared. By taking advantage of the excellent hydrophilicity and size-exclusion ability, we applied Fe3O4@mSiO2@G6P to capture glycopeptides from both horseradish peroxidase (HRP) and immunoglobulin (IgG) digests successfully. Moreover, a total of 39 and 25 endogenous glycopeptides were identified from healthy saliva and gastric saliva, respectively, indicating the great potential of this probe for the exploration of glycopeptidome biomarkers.

Fluorescence bioimaging potential, both in vitro and in vivo, of a yellow emissive triazole-based molecular marker has been investigated and demonstrated. Three different kinds of cells, viz Bacillus thuringiensis, Candida albicans, and Techoma stans pollen grains were used to investigate the intracellular zinc imaging potential of 1 (in vitro studies). Fluorescence imaging of translocation of zinc through the stem of small herb, Peperomia pellucida, having transparent stem proved in vivo bioimaging capability of 1. This approach will enable in screening cell permeability and biostability of a newly developed probe. Similarly, the current method for detection and localization of zinc in Gram seed sprouts could be an easy and potential alternative of the existing analytical methods to investigate the efficiency of various strategies applied for increasing zinc-content in cereal crops. The probe-zinc ensemble has efficiently been applied for detecting phosphate-based biomolecules Refereed/Peer-reviewed

Identifying cells associated with specific disease states is critically important for the early detection and diagnosis of cancer. To facilitate this task, molecular probes, which bind biomarkers that are either specifically or differentially expressed in diseased cells relative to healthy cells, provide a simple and effective method. This review focuses on the use of DNA aptamers as molecular probes for cancer cells. These aptamers are created by means of the cell-based Systematic Evolution of Ligands by EXponential enrichment (SELEX) process, which uses whole disease cells as targets. We describe at length the steps of the cell-SELEX process and discuss several applications for the aptamers, including profiling leukemia patient samples and discovering cell-surface cancer biomarkers. We conclude with a discussion of an aptamer-conjugated nanoparticle enrichment and detection scheme.

Focal adhesion kinase (FAK) has been identified as a potential therapeutic target for the treatment of metastatic cancers. Herein we describe the design, synthesis and optimization of a direct activity sensor for FAK and its application to screening FAK inhibitors. We find that the position of the sensing moiety, a phosphorylation-sensitive sulfonamido-oxine fluorophore, can dramatically influence the performance of peptide sensors for FAK. Real-time fluorescence activity assays using an optimized sensor construct, termed FAKtide-S2, are highly reproducible (Z' = 0.91) and are capable of detecting as little as 1 nM recombinant FAK. Utilizing this robust assay format, we define conditions for the screening of FAK inhibitors and demonstrate the utility of this platform using a set of well-characterized small molecule kinase inhibitors. Additionally, we provide the selectivity profile of FAKtide-S2 among a panel of closely related enzymes, identifying conditions for selectively monitoring FAK activity in the presence of off-target enzymes. In the long term, the chemosensor platform described in this work can be used to identify novel FAK inhibitor scaffolds and potentially assess the efficacy of FAK inhibitors in disease models.

DNA exhibits structural flexibility and may adopt also tetraplex structures known as guanine-quadruplexes or G-quadruplexes. These G-quadruplexes have recently received great attention because G-rich sequences are often found in genome and because of their potential links to mechanisms that relate to cancer, HIV, and other diseases. The unique structure of quadruplexes has also stimulated development of new analytical and bioanalytical assays based on fluorescence resonance energy transfer (FRET). Intramolecular folding of a flexible single-stranded DNA molecule into a compact G-quadruplex is a structural transition leading to closer proximity of its 5'- and 3'-ends. Thus, labeling both ends of a DNA strand with donor and acceptor fluorophores enables monitoring the quadruplex formation process by means of the FRET signal. This review shows how FRET technique contributes to G-quadruplex research and focuses mainly on analytical applications of FRET-labeled quadruplexes. Applications include studies of structural transitions of quadruplexes, FRET-based selection of ligands that bind to quadruplexes, design of molecular probes for protein recognition and development of sensors for detection of potassium ions in aqueous solution.

Force mapping with the atomic force microscope (AFM) allows the simultaneous acquisition of topography and probe–sample interaction data. For example, AFM probes functionalised with an antigen can be employed to map the spatial distribution of recognition events on a substrate functionalised with its specific antibody. However, to date this method has been limited to the detection of single receptor–ligand species. Were the detection of multiple receptor–ligand interactions possible, force mapping would offer great scope as a sensitive tool for bioassay and screening applications. We have developed an immobilisation strategy, which allows two different molecular species (in this case human serum albumin and the β subunit of human chorionic gonadotropin) to be present simultaneously on an AFM probe. Single point force spectroscopy results have revealed the ability of such probes to discriminate between their corresponding recognition points (anti-HSA and anti-βhCG IgG antibodies). As a control, force measurements were re-recorded in the presence of the known antigen (free in solution) for each antibody species and a marked decrease in the frequency of specific interaction is observed. As an additional control interactions between anti-βhCG IgG and the multifunctional probe are taken in the presence of free βhCG (“true” antigen) and free HSA (“false” antigen). It is shown that measurements recorded in the presence of a non-related protein species results in no change in either the force observed or the frequency of specific interactions, further confirmation that the specificity of force observed is due to the separation of antibody–antigen complex.

A novel assay for the voltammetric detection of DNA sequences related to the hepatitis B virus (HBV), using MB as the hybridization indicator was performed. The voltammetric signals of MB have been investigated at bare CPE, double stranded DNA (dsDNA)-modified CPE and single stranded DNA (ssDNA)-modified CPE by means of differential pulse voltammetry and cyclic voltammetry and the increased peak currents were observed, in respect to the order of electrodes. The extent of hybridization between the 21-mer oligonucleotides of complementary sequences was determined by the enhancement of the voltammetric signal of MB. The response of the hybridization of the probe with the single-base mismatch oligonucleotide at CPE was detected by MB. In this case, the unbound guanine bases increased the voltammetric signal obtained with the hybrid-modified CPE. Control experiments with the noncomplementary oligonucleotides were performed to assess whether the DNA biosensor responds selectively, via hybridization, to the target. Numerous factors, affecting the probe immobilization, target hybridization and indicator binding reactions are optimized to maximize the sensitivity and reduce the assay time. The new indicator MB holds great promise for rapid screening of HBV infection.

In this paper, we report the realisation of a DNA piezoelectric biosensor coupled with the polymerase chain reaction (PCR) for the detection of a specific bacterial toxicity factor. Biotinylated 23 ...

A new biosensing strategy for in situ electrochemical detection of DNA hybridization is described. The label-free approach relies on the doping of nucleic acid probes within electropolymerized polypyrrole (PPy) films and monitoring the current changes provoked by the hybridization event. The study demonstrates that oligonucleotide probes can serve as the sole counter anion during the growth of conducting PPy films, and maintain their hybridization activity within the host polymer network. Distinct hybridization peak signals, with opposite direction in the presence of complementary and non-complementary DNA sequences, are observed in real time. Such unique response is attributed to concomitant conductivity changes, and offers great promise for DNA diagnostics.

A chemiluminescence derivatization method using a novel reagent, 3′,4′,5′-trimethoxyphenylglyoxal (TMPG) was studied for the quantitative detection of nucleic acids and deoxyguanylic acid oligomers on a nylon membrane. In the method, TMPG rapidly converted guanine of a nucleic acid to a chemiluminescent derivative in an alkaline phosphate solution at a room temperature. A cooled, charge-coupled device camera was used to detect the chemiluminescence emitted at room temperature from the derivatives on the membrane after the TMPG reaction. The detection signal was increased by increasing the amount of deoxyguanylic acid in the analyte molecule. When a DNA probe having a 5′-(G) 15 TT(G) 15 TT deoxyoligomer at its 3′ terminus was hybridized with a target complementary DNA fragment on the membrane, an increased chemiluminescence from the hybrid was detected by the proposed method. The lower limit of detection is approximately 1.0 pmol of the target DNA fragment bound to the probe in the hybridization assay.

A new electrochemical hybridization biosensor protocol without an external indicator is described. The biosensor format involves the immobilization of inosine-substituted (guanine-free) probe onto the carbon paste transducer, and a direct chronopotentiometric detection of the duplex formation by the appearance of the guanine oxidation peak of the target. Such a use of the intrinsic DNA electrochemical response for monitoring hybridization events offers several advantages (over the common use of external indicators), including the appearance of a new peak, a flat background, or simplicity. A 4 min short hybridization period allows a detection limit around 120 ng/ml. Performance characteristics of the sensor are described along with future prospects.

The detection of higher-ordered sequences of DNA such as the dyad symmetrical sequence was investigated using Tb(III) luminescence, based on energy transfer from excited bases in a single stranded DNA probe. An oligonucleotide carrying an iminodiacetic acid function as metal chelating moiety was used as the probe. The Tb(III) complex with this probe gave luminescence characteristic of Tb(III). The intensity of this luminescence was considerably suppressed when bound to the targeted double stranded DNA to form a triple helix. This behavior was especially pronounced when the probe forms a 2:1 (probe strand: Tb(III)) complex on the target double strand carrying a dyad symmetrical base sequence. When the analyte DNA strand does not contain a complementary sequence to the probe, the intensity of the luminescence was scarcely influenced. This characteristic luminescence change will be useful for detecting target genes having a higher-ordered sequence.

An electrochemical biosensor for the determination of short sequences from the Mycobacterium tuberculosis (MTB) DNA is described. The sensor relies on the modification of the carbon-paste transducer with 27- or 36-mer oligonucleotide probes and their hybridization to complementary strands from the MTB DNA direct repeat region. Chronopotentiometry is employed to transduce the hybridization event, in connection with a Co(phen) 3 3+ indicator. Short (5–15 min) hybridization periods permit convenient quantitation of ng ml −1 levels of the MTB DNA target. Similar results are observed using microfabricated carbon-strip transducers. The new sensor holds great promise for rapid screening for MTB infections. Direct adsorptive stripping potentiometric measurements of ultratrace levels of MTB DNA are also reported.

As promising molecular probes for in vivo tumor imaging, aptamers without modification remain problematic due to insufficient serum stability and unabiding imaging window. To address this problem, a novel locked nucleic acid (LNA)/DNA chimeric aptamer probe was developed through proper LNA incorporation and supplemented 3'-3'-thymidine (3'-3'-T) capping. TD05, a DNA aptamer against lymphoma Ramos cells, being used as the model, a series of modification strategies were designed and optimized with different positions, numbers and combinations. It was revealed that the combined use of LNA and 3'-3'-T had a synergistic effect, and with the increase of LNA substitution in stem region, the serum stability of TD05 was gradually enhanced while its affinity and specificity were perfectly maintained to Ramos cells. Particularly, TD05.6 with 7-base pair-LNA substitution exhibited the significantly elevated detection stability half-life from ∼0.5 h of TD05 to 5-6h of TD05.6 for target cells in serum. Moreover, a much slower clearance rate in tumor-bearing mice was also observed for TD05.6, thus leading to the greatly extended tumor imaging window from150 min of TD05 to600 min of TD05.6. This strategy might be of great potentials to generate more aptamer probes that are stable and nuclease-resistant for tumor diagnosis in real biological systems.

Although pyrene is a popular fluorescent molecular probe in analytical and bioanalytical chemistry, its photostability is hardly understood. This paper reports a surprising finding that pyrene undergoes significant photochemical reactions in alcohol and aqueous solvents under the condition of exposing a sample solution to excitation irradiation in an emission spectrometer for a short period of time. The reactions yield at least two photoproducts, whose fluorescence spectra overlap severely with that of pyrene. Implications of pyrene photochemical reactions in alcohol and aqueous solvent systems to existing and future applications of pyrene as a fluorescent molecular probe are discussed.

The usefulness of principal component analysis (PCA) and factor analysis (FA) for source input elucidation in environmental studies using molecular markers for sample description was evaluated. A case study involving the determination of aliphatic and chlorinated hydrocarbons, fatty acids, alcohols, chlorophylls and some detergent indicators in water particulates from a deltaic system was selected as a representative testing dataset. PCA afforded useful results to differentiate between major groups of samples but not between geochemical sources. In contrast, FA provided a direct correspondence between factor loadings and marker groups defining geochemically consistent organic matter contributions. For autochthonous compounds, FA has allowed an even more precise characterization of input sources than that obtained by the common “qualitative” molecular marker approach. These results were obtained despite the unsuitable dimensions of the dataset describing the system [102 compounds (variables) × 40 samples]. No improved results were obtained from the application of FA or PCA to subgroups of the data involving smaller number of compounds. This is consistent with the hypothesis that a limited number of sources is responsible for the occurrence of these lipid markers in the delta waters.