DNA-binding small-ligand-immobilized surface plasmon resonance biosensor for detecting thymine-related single-nucleotide polymorphisms

A surface plasmon resonance (SPR) biosensor that carries DNA-binding small ligands has been developed for the detection of single-nucleotide polymorphisms (SNPs). 3,5-Diaminopyrazine derivatives, with a hydrogen-bonding profile fully complementary to the thymine base, were utilized as recognition elements on the sensor surface, and a target single-stranded DNA sequence was hybridized with a DNA probe containing an abasic site to place this site opposite a nucleobase to be detected. In a continuous flow of sample solutions buffered to pH 6.4 (0.25 M NaCl), the 3,5-diaminopyrazine-based SPR sensor can detect an orphan nucleobase in the duplex with a clear selectivity for thymine over cytosine, guanine, and adenine (5'-GTT GGA GCT GXG GGC GTA GGC-3'/3'-CAA CCT CGA CNC CCG CAT CCG-5'; X=abasic site, N=target nucleobase G, C, A, or T). The SPR response was linear in the concentration range 10-100 nM. Allele discrimination is possible based on the combination of different binding surfaces in a flow cell of the SPR system, which is demonstrated for the analysis of the thymine/cytosine mutation present in 63-meric polymerase chain reaction (PCR) amplification products (Ha-ras gene, codon 12, antisense strand). Comparison with a bulk assay based on 3,5-diaminopyrazine/DNA binding shows that the immobilization of 3,5-diaminopyrazine derivatives on the SPR sensor allows more sensitive detection of the target DNA sequence, and binding selectivity can be tuned by controlling the salt concentration of sample solutions. These features of the DNA-binding small-molecule-immobilized SPR sensor are discussed as a basis for the design of SPR biosensors for SNP genotyping.     

Sensing Enzymatic Activity by Exposure and Selection of DNA‐Encoded Probes

A sensing approach is applied to encode quantitative enzymatic activity information into DNA sequence populations. The method utilizes DNA‐linked peptide substrates as activity probes. Signal detection involves chemical manipulation of a probe population downstream of sample exposure and application of purifying, selective pressure for enzyme products. Selection‐induced changes in DNA abundance indicate sample activity. The detection of protein kinase, protease, and farnesyltransferase activities is demonstrated. The assays were employed to measure enzyme inhibition by small molecules and activity in cell lysates using parallel DNA sequencing or quantitative PCR. This strategy will allow the extensive infrastructure for genetic analysis to be applied to proteomic assays, which has a number of advantages in throughput, sensitivity, and sample multiplexing.     

Assembling DNA through Affinity Binding to Achieve Ultrasensitive Protein Detection

Recent advances in DNA assembly and affinity binding have enabled exciting developments of nanosensors and ultrasensitive assays for specific proteins. 1 – 6 These sensors and assays share three main attractive features: 1 , 4 , 7 1) the detection of proteins can be accomplished by the detection of amplifiable DNA, thereby dramatically enhancing the sensitivity; 2) assembly of DNA is triggered by affinity binding of two or more probes to a single target molecule, thereby resulting in increased specificity; and 3) the assay is conducted in solution with no need for separation, thus making the assay attractive for potential point‐of‐care applications. We illustrate here the principle of assembling DNA through affinity binding, and we highlight novel applications to the detection of proteins.     

Rational Design of Carboxyl Groups Perpendicularly Attached to a Graphene Sheet: A Platform for Enhanced Biosensing Applications

Graphene oxide (GO)‐based materials offer great potential for biofunctionalization with applications ranging from biosensing to drug delivery. Such biofunctionalization utilizes specific functional groups, typically a carboxyl moiety, as anchoring points for biomolecule. However, due to the fact that the exact chemical structure of GO is still largely unknown and poorly defined (it was postulated to consist of various oxygen‐containing groups, such as epoxy, hydroxyl, carboxyl, carbonyl, and peroxy in varying ratios), it is challenging to fabricate highly biofunctionalized GO surfaces. The predominant anchoring sites (i.e., carboxyl groups) are mainly present as terminal groups on the edges of GO sheets and thus account for only a fraction of the oxygen‐containing groups on GO. Herein, we suggest a direct solution to the long‐standing problem of limited abundance of carboxyl groups on GO; GO was first reduced to graphene and consequently modified with only carboxyl groups grafted perpendicularly to its surface by a rational synthesis using free‐radical addition of isobutyronitrile with subsequent hydrolysis. Such grafted graphene oxide can contain a high amount of carboxyl groups for consequent biofunctionalization, at which the extent of grafting is limited only by the number of carbon atoms in the graphene plane; in contrast, the abundance of carboxyl groups on “classical” GO is limited by the amount of terminal carbon atoms. Such a graphene platform embedded with perpendicularly grafted carboxyl groups was characterized in detail by X‐ray photoelectron spectroscopy, cyclic voltammetry, and electrochemical impedance spectroscopy, and its application was exemplified with single‐nucleotide polymorphism detection. It was found that the removal of oxygen functionalities after the chemical reduction enhanced the electron‐transfer rate of the graphene. More importantly, the introduction of carboxyl groups promoted a more efficient immobilization of DNA probes on the electrode surface and improved the performance of graphene as a biosensor in comparison to GO. The proposed material can be used as a universal platform for biomolecule immobilization to facilitate rapid and sensitive detection of DNA or proteins for point‐of‐care investigations. Such reactive carboxyl groups grafted perpendicularly on GO holds promise for a highly efficient tailored biofunctionalization for applications in biosensing or drug delivery.     

Trends and perspectives in DNA biosensors as diagnostic devices

Despite the civilization and technological development, taking care of health based on early diagnostics is still challenging. Currently, cancer accounts for more than 20% of all deaths. Cancer mortality dramatically rises every year because of poor diagnosis at the late stage and inefficiency of conventional methods for early-stage cancer detection. That is why there is a demand for automated, inexpensive, miniaturized, and portable testing devices with real-time response, high sensitivity, and selectivity for early medical diagnostics but also for screening air and water. DNA biosensors have excellent predispositions and are a significant promise to become a powerful tool used in prevention and monitoring of diseases, rationalization of the way of medical treatment, and improving the patient quality of life.     

Nucleic acid functionalized graphene for biosensing

There is immense demand for complex nanoarchitectures based on graphene nanostructures in the fields of biosensing or nanoelectronics. DNA molecules represent the most versatile and programmable recognition element and can provide a unique massive parallel assembly strategy with graphene nanomaterials. Here we demonstrate a facile strategy for covalent linking of single stranded DNA (ssDNA) to graphene using carbodiimide chemistry and apply it to genosensing. Since graphenes can be prepared by different methods and can contain various oxygen containing groups, we thoroughly investigated the utility of four different chemically modified graphenes for functionalization by ssDNA. The materials were characterized in detail and the different DNA functionalized graphene platforms were then employed for the detection of DNA hybridization and DNA polymorphism by using impedimetric methods. We believe that our findings are very important for the development of novel devices that can be used as alternatives to classical techniques for sensitive and fast DNA analysis. In addition, covalent functionalization of graphene with ssDNA is expected to have broad implications, from biosensing to nanoelectronics and directed, DNA programmable, self-assembly.     

Two-temperature hybridization for microarray detection of label-free microRNAs with attomole detection and superior specificity

Two is better than one: Two short locked nucleic acid based probes were used to collectively capture and detect microRNAs by a simple two-temperature hybridization process. Intact microRNAs were directly measured down to attomolar concentrations with a high specificity and nearly four orders of magnitude of dynamic range. Single base mismatches in the microRNAs were potently discriminated from the perfectly matched targets.     

Development of fluorescent glucose bioprobes and their application on real-time and quantitative monitoring of glucose uptake in living cells

We developed a novel fluorescent glucose bioprobe, GB2-Cy3, for the real-time and quantitative monitoring of glucose uptake in living cells. We synthesized a series of fluorescent glucose analogues by adding Cy3 fluorophores to the α-anomeric position of D-glucose through various linkers. Systematic and quantitative analysis of these Cy3-labeled glucose analogues revealed that GB2-Cy3 was the ideal fluorescent glucose bioprobe. The cellular uptake of this probe competed with the cellular uptake of D-glucose in the media and was mediated by a glucose-specific transport system, and not by passive diffusion. Flow cytometry and fluorescence microscopy analyses revealed that GB2-Cy3 is ten times more sensitive than 2-NBDG, a leading fluorescent glucose bioprobe. GB2-Cy3 can also be utilized for the quantitative flow cytometry monitoring of glucose uptake in metabolically active C2C12 myocytes under various treatment conditions. As opposed to a glucose uptake assay performed by using radioisotope-labeled deoxy-D-glucose and a scintillation counter, GB2-Cy3 allows the real-time monitoring of glucose uptake in living cells under various experimental conditions by using fluorescence microscopy or confocal laser scanning microscopy (CLSM). Therefore, we believe that GB2-Cy3 can be utilized in high-content screening (HCS) for the discovery of novel therapeutic agents and for making significant advances in biomedical studies and diagnosis of various diseases, especially metabolic diseases.