Amir Lichtenstein

Detection of single-base DNA mutations by enzyme-amplified electronic transduction

Here we describe a method for the sensitive detection of a single-base mutation in DNA. We assembled a primer thiolated oligonucleotide, complementary to the target DNA as far as one base before the mutation site, on an electrode or a gold–quartz piezoelectric crystal. After hybridizing the target DNA, normal or mutant, with the sensing oligonucleotide, the resulting assembly is reacted with the biotinylated nucleotide, complementary to the mutation site, in the presence of polymerase. The labeled nucleotide is coupled only to the double-stranded assembly that includes the mutant site. Subsequent binding of avidin–alkaline phosphatase to the assembly, and the biocatalyzed precipitation of an insoluble product on the transducer, provides a means to confirm and amplify detection of the mutant. Faradaic impedance spectroscopy and microgravimetric quartz-crystal microbalance analyses were employed for electronic detection of single-base mutants. The lower limit of sensitivity for the detection of the mutant DNA is 1 × 10−14 mol/ml. We applied the method for the analysis of polymorphic blood samples that include the Tay–Sachs genetic disorder. The sensitivity of the method enables the quantitative analysis of the mutant with no PCR pre-amplification.

Enzyme monolayer-functionalized field-effect transistors for biosensor applications

Abstract A gate surface of an ion-selective field-effect transistor was modified with a monolayer enzyme array that stimulates biocatalytic reactions that control the gate potential. Stepwise assemblage of the biocatalytic layer included primary silanization of the Al 2 O 3 -gate with 3-aminopropyltriethoxysilane, subsequent activation of the amino groups with glutaric dialdehyde and the covalent attachment of the enzyme to the functionalized gate surface. Urease, glucose oxidase, acetylcholine esterase and α-chymotrypsin were used to organize the biocatalytic matrices onto the chip gate. The resulting enzyme-based field-effect transistors, ENFETs, demonstrated capability to sense urea, glucose, acetylcholine and N -acetyl- l -tyrosine ethyl ester, respectively. The mechanism of the biosensing involves the alteration of the pH in the sensing layer by the biocatalytic reactions and the detection of the pH change by the ENFET. The major advantage of the enzyme-thin-layered FET devices as biosensors is the fast response-time (several tens of seconds) of these bioelectronic devices. This advantage over traditional thick-polymer-based ENFETs results from the low diffusion barrier for the substrate penetration to the biocatalytic active sites and minute isolation of the pH-sensitive gate surface from the bulk solution.

Biorecognition Layer Engineering: Overcoming Screening Limitations of Nanowire-Based FET Devices

Detection of biological species is of great importance to numerous areas of medical and life sciences from the diagnosis of diseases to the discovery of new drugs. Essential to the detection mechanism is the transduction of a signal associated with the specific recognition of biomolecules of interest. Nanowire-based electrical devices have been demonstrated as a powerful sensing platform for the highly sensitive detection of a wide-range of biological and chemical species. Yet, detecting biomolecules in complex biosamples of high ionic strength (>100 mM) is severely hampered by ionic screening effects. As a consequence, most of existing nanowire sensors operate under low ionic strength conditions, requiring ex situ biosample manipulation steps, that is, desalting processes. Here, we demonstrate an effective approach for the direct detection of biomolecules in untreated serum, based on the fragmentation of antibody-capturing units. Size-reduced antibody fragments permit the biorecognition event to occur in closer proximity to the nanowire surface, falling within the charge-sensitive Debye screening length. Furthermore, we explored the effect of antibody surface coverage on the resulting detection sensitivity limit under the high ionic strength conditions tested and found that lower antibody surface densities, in contrary to high antibody surface coverage, leads to devices of greater sensitivities. Thus, the direct and sensitive detection of proteins in untreated serum and blood samples was effectively performed down to the sub-pM concentration range without the requirement of biosamples manipulation.

Si nanowires forest-based on-chip biomolecular filtering, separation and preconcentration devices: nanowires do it all.

The development of efficient biomolecular separation and purification techniques is of critical importance in modern genomics, proteomics, and biosensing areas, primarily due to the fact that most biosamples are mixtures of high diversity and complexity. Most of existent techniques lack the capability to rapidly and selectively separate and concentrate specific target proteins from a complex biosample, and are difficult to integrate with lab-on-a-chip sensing devices. Here, we demonstrate the development of an on-chip all-SiNW filtering, selective separation, desalting, and preconcentration platform for the direct analysis of whole blood and other complex biosamples. The separation of required protein analytes from raw biosamples is first performed using a antibody-modified roughness-controlled SiNWs (silicon nanowires) forest of ultralarge binding surface area, followed by the release of target proteins in a controlled liquid media, and their subsequent detection by supersensitive SiNW-based FETs arrays fabricated on the same chip platform. Importantly, this is the first demonstration of an all-NWs device for the whole direct analysis of blood samples on a single chip, able to selectively collect and separate specific low abundant proteins, while easily removing unwanted blood components (proteins, cells) and achieving desalting effects, without the requirement of time-consuming centrifugation steps, the use of desalting or affinity columns. Futhermore, we have demonstrated the use of our nanowire forest-based separation device, integrated in a single platform with downstream SiNW-based sensors arrays, for the real-time ultrasensitive detection of protein biomarkers directly from blood samples. The whole ultrasensitive protein label-free analysis process can be practically performed in less than 10 min.

Supersensitive fingerprinting of explosives by chemically modified nanosensors arrays

The capability to detect traces of explosives sensitively, selectively and rapidly could be of great benefit for applications relating to civilian national security and military needs. Here, we show that, when chemically modified in a multiplexed mode, nanoelectrical devices arrays enable the supersensitive discriminative detection of explosive species. The fingerprinting of explosives is achieved by pattern recognizing the inherent kinetics, and thermodynamics, of interaction between the chemically modified nanosensors array and the molecular analytes under test. This platform allows for the rapid detection of explosives, from air collected samples, down to the parts-per-quadrillion concentration range, and represents the first nanotechnology-inspired demonstration on the selective supersensitive detection of explosives, including the nitro- and peroxide-derivatives, on a single electronic platform. Furthermore, the ultrahigh sensitivity displayed by our platform may allow the remote detection of various explosives, a task unachieved by existing detection technologies.

Dendritic amplification of DNA analysis by oligonucleotide-functionalized Au-nanoparticles

Dendritic amplification of DNA analysis is accomplished by the application of 5′- and 3′-terminated oligonucleotide-functionalized Au-colloids complementary to the analyte DNA.

Non-covalent Monolayer-Piercing Anchoring of Lipophilic Nucleic Acids: Preparation, Characterization, and Sensing Applications

Functional interfaces of biomolecules and inorganic substrates like semiconductor materials are of utmost importance for the development of highly sensitive biosensors and microarray technology. However, there is still a lot of room for improving the techniques for immobilization of biomolecules, in particular nucleic acids and proteins. Conventional anchoring strategies rely on attaching biomacromolecules via complementary functional groups, appropriate bifunctional linker molecules, or non-covalent immobilization via electrostatic interactions. In this work, we demonstrate a facile, new, and general method for the reversible non-covalent attachment of amphiphilic DNA probes containing hydrophobic units attached to the nucleobases (lipid-DNA) onto SAM-modified gold electrodes, silicon semiconductor surfaces, and glass substrates. We show the anchoring of well-defined amounts of lipid-DNA onto the surface by insertion of their lipid tails into the hydrophobic monolayer structure. The surface coverage of DNA molecules can be conveniently controlled by modulating the initial concentration and incubation time. Further control over the DNA layer is afforded by the additional external stimulus of temperature. Heating the DNA-modified surfaces at temperatures >80 °C leads to the release of the lipid-DNA structures from the surface without harming the integrity of the hydrophobic SAMs. These supramolecular DNA layers can be further tuned by anchoring onto a mixed SAM containing hydrophobic molecules of different lengths, rather than a homogeneous SAM. Immobilization of lipid-DNA on such SAMs has revealed that the surface density of DNA probes is highly dependent on the composition of the surface layer and the structure of the lipid-DNA. The formation of the lipid-DNA sensing layers was monitored and characterized by numerous techniques including X-ray photoelectron spectroscopy, quartz crystal microbalance, ellipsometry, contact angle measurements, atomic force microscopy, and confocal fluorescence imaging. Finally, this new DNA modification strategy was applied for the sensing of target DNAs using silicon-nanowire field-effect transistor device arrays, showing a high degree of specificity toward the complementary DNA target, as well as single-base mismatch selectivity.

Liposome-encapsulated silver sulfadiazine (SSD) for the topical treatment of infected burns: Thermodynamics of drug encapsulation and kinetics of drug release

Liposomes encapsulating silver sulfadiazine (SSD), the drug of choice for topical treatment of infected burns, are investigated as an improved delivery system that could act as a locally targeted sustained-release drug depot. This communication reports the first stage of the investigation and is focused on (a) the development of spectrophotometric assays for liposome-encapsulated and for free (aqueous soluble) and SSD, (b) on evaluation of the efficiency of encapsulation and kinetics of drug release. DMSO containing 140 mM NH3 was found to be the best solvent for dissolution of the liposomes and for determination of their SSD content. Peak absorption of liposome-originating SSD in this solvent is at 263 nm with em values of 23 x 10(3)-26 x 10(3). Peak absorption of SSD in aqueous solutions is at 254 nm with em magnitudes varying from 2 x 10(3) to 23 x 10(3), depending on the electrolytic composition of the system. Kinetic studies of drug release and separations by centrifugation and by gel-exclusion chromatography all indicate that the SSD in the liposomal system is distributed among three states: encapsulated, soluble unencapsulated, and stable (unencapsulated) aggregates that reside in the aqueous phase in which the liposomes are suspended. The liposomal SSD systems were found to meet the essential requirements of high-efficiency encapsulation and sustained drug release. Encapsulation efficiencies of > 80% at 10 mM lipid, reaching up to 95% at 100 mM lipid, were obtained. The release of encapsulated SSD follows first-order kinetics, with half-life up to 24 hr and with sensitivity to the electrolytes in the system. It is concluded that SSD-liposomal systems are feasible, have potential benefits over treatment with free SSD, and merit further pursuit into providing local targeting.

Ultrasensitive and Specific Electronic Transduction of DNA Sensing Processes

Amplified electronic transduction of DNA sensing is accomplished by the application of a biocatalytic probe that precipitates an insoluble product on the transducer as a result of the DNA sensing. Alternative amplification of DNA sensing is achieved by the use of functionalized liposomes that bind to the electrode upon the sensing process. Single base mutations in DNA are detected by the polymerase-induced coupling of a biotinylated base complementary to the mismatch site, followed by the biocatalyzed precipitation of an insoluble product on the transducer. DNA biosensors of unprecedented sensitivity and specificity are designed. Faradaic impedance spectroscopy and microgravimetric quartz-crystal-microbalance measurements are used for the electronic transduction of the DNA sensing.