PNA biosensors for nucleic acid detection

Biosensor devices, based on the conversion of nucleic acid recognition reactions into useful electrical signals, offer considerable promise for DNA diagnostics. The unique hybridization properties of solution-phase PNA can be extrapolated onto transducer surfaces in connection with the design of remarkably specific DNA biosensors. This article reviews the development of PNA biosensors, and discusses common PNA-biosensing protocols along with their prospects in DNA biosensor technology.     

Electrochemical biosensors: towards point-of-care cancer diagnostics

Wide-scale point-of-care diagnostic systems hold great promise for early detection of cancer at a curable stage of the disease. This review discusses the prospects and challenges of electrochemical biosensors for next-generation cancer diagnostics. Electrochemical biosensors have played an important significant role in the transition towards point-of-care diagnostic devices. Such electrical devices are extremely useful for delivering the diagnostic information in a fast, simple, and low cost fashion in connection to compact (hand-held) analyzers. Modern electrochemical bioaffinity sensors, such as DNA- or immunosensors, offer remarkable sensitivity essential for early cancer detection. The coupling of electrochemical devices with nanoscale materials offers a unique multiplexing capability for simultaneous measurements of multiple cancer markers. The attractive properties of electrochemical devices are extremely promising for improving the efficiency of cancer diagnostics and therapy monitoring. With further development and resources, such portable devices are expected to speed up the diagnosis of cancer, making analytical results available at patient bedside or physician office within few minutes.     

Ultrasensitive electrical detection of nucleic acids by hematin catalysed silver nanoparticle formation in sub-microgapped biosensors

An ultrasensitive electrical detection method of nucleic acids has been demonstrated on sub-microgapped biosensor. In this method, peptide nucleic acid (PNA) probes were firstly immobilized in the gap areas of a pair of interdigited microelectrodes and then were hybridized with their complementary target DNA. After hybridization, hematin molecules were introduced into the DNA strand via zirconium-phosphate and zirconium-carbonate chemistries. The newly attached hematin molecules act as a catalyst to accelerate reducing ammoniacal silver ion to form silver nanoparticles, which span the gap of the interdigitated microelectrode. The conductance of the silver nanoparticles directly correlated with the number of the hybridized DNA molecules. Nearly 1fM sensitivity was achieved under optimal conditions. This approach is also applicable to the detection of RNA.     

基于CRISPR/Cas体系的生物传感器在病原体核酸检测方面的应用

成簇规律间隔短回文重复序列/成簇规律间隔短回文重复序列相关蛋白 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein,CRISPR/Cas) 因高效的靶向结合和可编程性,已被开发为一种精准、高效、低价和高灵敏度的核酸检测工具。目前基于CRISPR-Cas体系的生物传感器在病原体核酸检测方面显示出了优良的性能,受到了人们的广泛关注,这种新型的病原体核酸检测有望替代传统的病原体检测方法。文中就基于CRISPR/Cas体系的生物传感器在病原体核酸检测中的最新研究进展进行综述。     

Microfluidic-integrated biosensors: Prospects for point-of-care diagnostics

There is a growing demand to integrate biosensors with microfluidics to provide miniaturized platforms with many favorable properties, such as reduced sample volume, decreased processing time, low cost analysis and low reagent consumption. These microfluidics-integrated biosensors would also have numerous advantages such as laminar flow, minimal handling of hazardous materials, multiple sample detection in parallel, portability and versatility in design. Microfluidics involves the science and technology of manipulation of fluids at the micro- to nano-liter level. It is predicted that combining biosensors with microfluidic chips will yield enhanced analytical capability, and widen the possibilities for applications in clinical diagnostics. The recent developments in microfluidics have helped researchers working in industries and educational institutes to adopt some of these platforms for point-of-care (POC) diagnostics. This review focuses on the latest advancements in the fields of microfluidic biosensing technologies, and on the challenges and possible solutions for translation of this technology for POC diagnostic applications. We also discuss the fabrication techniques required for developing microfluidic-integrated biosensors, recently reported biomarkers, and the prospects of POC diagnostics in the medical industry.     

Ultrasensitive carbon nanotube-based biosensors using antibody-binding fragments

We report a method to build ultrasensitive carbon nanotube-based biosensors using immune binding reaction. Here carbon nanotube-field effect transistors (CNT-FETs) were functionalized with antibody-binding fragments as a receptor, and the binding event of target immunoglobulin G (IgG) onto the fragments was detected by monitoring the gating effect caused by the charges of the target IgG. Because the biosensors were used in buffer solution, it was crucial to use small-size receptors so that the charged target IgG could approach the CNT surface within the Debye length distance to give a large gating effect. The results show that CNT-FET biosensors using whole antibody had very low sensitivity (detection limit ∼1000 ng/ml), whereas those based on small Fab fragments could detect 1 pg/ml (∼7 fM level). Moreover, our Fab-modified CNT-FET could successfully block the nontarget proteins and could selectively detect the target protein in an environment similar to that of human serum electrolyte. Significantly, this strategy can be applied to general antibody-based detection schemes, and it should enable the production of label-free ultrasensitive electronic biosensors to detect clinically important biomarkers for disease diagnosis.     

Computational selection of nucleic acid biosensors via a slip structure model

Aptamers have been shown to undergo ligand-dependent conformational changes, and can be joined to ribozymes to create allosteric ribozymes (aptazymes). An anti-flavin (FMN) aptamer joined to the hammerhead ribozyme yielded an aptazyme that underwent small, FMN-dependent displacements in the helix that joined the aptamer and ribozyme. This 'slip structure' model in which alternative sets of base-pairs are formed in the absence and presence of ligand proved amenable to energetic and computational modeling. Initial successes in modeling the activities of known aptazymes led to the in silico selection of new ligand-dependent aptazymes from virtual pools that contained millions of members. Those aptazymes that were predicted to best fit the slip structure model were synthesized and assayed, and the best-designed aptazyme was activated 60-fold by FMN. The slip structure model proved to be generalizable, and could be applied with equal facility to computationally generate aptazymes that proved to be experimentally activated by other ligands (theophylline) or that contained other catalytic cores (hairpin ribozyme). Moreover, the slip structure model could be applied to the prediction of a ligand-dependent aptamer beacon biosensor in which the addition of the protein vascular endothelial growth factor (VegF) led to a 10-fold increase in fluorescent signal.     

Ultrasensitive staining of nucleic acids with silver

A method for ultrasensitive detection of proteins on polyacrylamide gels by staining with silver, recently described by C. R. Merril, D. Goldman, S. A. Sedman, and M. H. Ebert (Science211, 1437–1438 (1981)), was applied with slight modifications to staining nucleic acids. Silver staining of double-stranded DNA was at least 100 times as sensitive as fluorescence staining with ethidium bromide, and at least 20 times as sensitive as staining with ammoniacal silver. The limit of detection of double-stranded DNA was approximately 25–50 pg/band with a cross-sectional area of 5 mm². The intensities of silver staining of double-stranded fragments 271 bp or longer from HaeIII endonuclease digests of φX174 RF DNA were linear over a concentration range of 0.25 to 4 ng DNA/band. RNA and single-stranded DNA species as short as 10 to 20 nucleotides were detected with high sensitivity after electrophoresis on denaturing gels containing urea, suggesting that silver staining may be applicable to the sequencing of a few micrograms of unlabeled DNA. Methods for staining DNA using ammoniacal silver were relatively insensitive for small DNA fragments.     

Design and synthesis of polyacrylamide-based oligonucleotide supports for use in nucleic acid diagnostics.

Polyacrylamide supports, in a range of pore sizes, were investigated as nucleic acid affinity matrices for the detection of target DNA or RNA sequences using a sandwich hybridization format. Bromoacetyl and thiol oligonucleotide derivatives were covalently linked to sulfhydryl- and bromoacetyl-polyacrylamide supports with greater than 95% end-attachment efficiencies. These polyacrylamide-oligonucleotide supports were further derivatized with anionic residues to provide multi-functional supports which show low non-specific binding for non-complementary nucleic acids. While all the polyacrylamide-oligonucleotide supports capture complementary oligonucleotides with high affinity, the pore size was found to be a critical parameter in sandwich hybridization reactions. The superior hybridization characteristics of the Trisacryl support was ascribed to a combination of its macroporous nature, hydrophilicity and the terminal attachment of its capture oligonucleotides.     

SELDI-TOF-based serum proteomic pattern diagnostics for early detection of cancer

Proteomics is more than just generating lists of proteins that increase or decrease in expression as a cause or consequence of pathology. The goal should be to characterize the information flow through the intercellular protein circuitry that communicates with the extracellular microenvironment and then ultimately to the serum/plasma macroenvironment. The nature of this information can be a cause, or a consequence, of disease and toxicity-based processes. Serum proteomic pattern diagnostics is a new type of proteomic platform in which patterns of proteomic signatures from high dimensional mass spectrometry data are used as a diagnostic classifier. This approach has recently shown tremendous promise in the detection of early-stage cancers. The biomarkers found by SELDI-TOF-based pattern recognition analysis are mostly low molecular weight fragments produced at the specific tumor microenvironment.     

Label‐free indicator‐free nucleic acid biosensors using carbon nanotubes

Carbon nanotubes (CNTs) are promising components for electrical biosensors due to their high surface‐to‐volume ratio and improved electron transfer properties. This review surveys CNT‐based label‐free indicator‐free biosensing strategies that have been demonstrated for the sensitive detection of nucleic acids. After an introduction to CNTs, the fabrication of biosensors and techniques for the immobilization of probe nucleic acids are outlined. Subsequently, two major label‐free strategies namely electrochemical transduction and field‐effect detection are presented. The focus is on direct detection methods that avoid labels, indicators, intercalating agents, mediators, and even secondary receptors. The review concludes with a comparison between the various biosensors and presents ways of engineering them so that they can be deployed in realistic diagnostic applications.     

A multidisciplinary approach for molecular diagnostics based on biosensors and microarrays

The present review describes the multidisciplinary approach followed by the Chemistry and Nanobiotechnology group of INL since ten years, to develop a complete technological platform dedicated to molecular diagnosis using biochips and biosensors. This work, replaced in an international context, illustrates the importance to identify the various pitfalls inherent to molecular analysis throughout an elaboration and analysis line: choice of solid support, surface physicochemistry, immobilisation of biomolecules, biomolecular recognition, microfluidics and detection.     

Peptide nucleic acid: a versatile tool in genetic diagnostics and molecular biology

During the past ten years, the DNA mimic peptide nucleic acid has inspired the development of a variety of hybridisation-based methods for detection, quantification, purification and characterisation of nucleic acids. Most of these methods have taken advantage of the very favourable DNA and RNA hybridisation properties of peptide nucleic acids combined with the unique properties and opportunities offered by peptide chemistry. Within the past year, significant progress in in situ hybridisation technology has been achieved, which has resulted, in particular, in reliable and sensitive methods for detection of bacteria in clinical samples, as well as in environmental samples. Furthermore, applications of the polymerase chain reaction clamping method have been expanded, and novel ways of exploiting complexes of peptide nucleic acids with double-stranded DNA, such as double duplex invasion complexes and PD loops, have been developed.     

Peptide nucleic acids on microarrays and other biosensors

The analysis of biomolecules using microarrays and other biosensors has a significant role in molecular biotechnology, and will become even more important in the future as a versatile tool for research and diagnostics. For many applications, the synthetic DNA mimic peptide nucleic acid (PNA) could be advantageous as a probe molecule, owing to its unique physicochemical and biochemical properties. PNA exhibits superior hybridization characteristics and improved chemical and enzymatic stability relative to nucleic acids. Furthermore, its different molecular structure enables new modes of detection, especially procedures that avoid the introduction of a label. In our opinion, all of these factors contribute significantly toward the establishment of faster and more reliable analytical processes and opens new fields of application.     

Piezoelectric biosensors: strategies for coupling nucleic acids to piezoelectric devices

The development of a piezoelectric biosensor based on nucleic acids interaction is presented focusing on the methodology for probe immobilization. This is a key step in any DNA biosensor development. Often, the detection limits and, in general, the analytical performances of the biosensor can be improved by optimizing the immobilization of the receptor on the transducer surface. DNA must be attached to the solid support, retaining native conformation, and binding activity. This attachment must be stable over the course of a binding assay and, in addition, sufficient binding sites must be presented to the solution phase to interact with the analyte. In this paper, the optimization of the coating of the gold quartz crystal surface, to immobilize an oligonucleotide probe, is reported. Two immobilization procedures are illustrated in details with a comparison regarding the immobilization of the probe, the detection of the hybridization reaction, and the possibility of regeneration. The two procedures are based on the use of biotinylated or thiolated DNA probes. Specific applications will be also presented.