Fernando Patolsky

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.

Au-Nanoparticle Nanowires Based on DNA and Polylysine Templates

The assembly of ordered nanoparticle architectures is a challenging topic in nanotechnology directed to the construction of nanoscale devices.[1] Within this broad subject, the conjugation of biomaterials and nanoparticles to yield ordered architectures is a promising route to tailor future sensing and catalytic devices, nanocircuitry, or nanodevices, for example transistors, and computing devices.[2] DNA is an attractive biomaterial for use as a template in programmed nanoparticle structures. The ability to synthesize nucleic acids of predesigned shapes and composition, the versatile biocatalytic transformations that can be performed on DNA, for example, ligation, scission, or polymerization, enable TMcut and paste∫ procedures to be carried out on the template DNA, thus enabling us to design and manipulate the DNA ∫mold∫. Furthermore, the association of metal ions to the DNA phosphate units, or the intercalation of transition-metal complexes or molecular substrates into the DNA provide a means to functionalize the DNA-template and to initiate further chemical transformations on the mold. Nanoparticle ± DNA assemblies were organized by the hybridization of nucleic-acid-functionalized metal[3] or semiconductor nanoThe fluorescence data in our experiments indicated that the surface coverage of the final printed layer for each of the three patterning methods presented here is nearly equivalent and reaches about 60% of the surface coverage obtained by direct deposition of the antibodies from solution. As already described for CP and CP of proteins, the printing process does not compromise the binding efficiency of the printed antibody. This strategy might not be suitable for patterning a large number of different proteins on a surface. However, it can place a few different proteins as adjacent high-density arrays on a surface. Such arrays could find an application for high-throughput screening in which a large number of analytes could be spotted using a subset of the patterned areas. Another possibility for creating high-density immunoassays on planar surfaces is by performing surface immunoassays using many different analytes and capture sites, such as shown in Figure 5. The main limiting factor in using the prepared microarrays for diagnostic purposes could be misplacement of target molecules during the inking of the stamp. Such a misplacement, which may induce false positive reactions, can arise from cross-reactions of the target molecules with different capture proteins and/or from nonspecific adsorption on the -stamp. The former is limited by biological specificity of affinity extraction. The latter can be limited by the systematic use of blocking agents such as BSA. Indeed, for the recognition of goat antigen by the printed array shown in Figure 5a, the recognition signal in the areas with printed anti-chicken antibodies was only 5% of that in the areas with printed anti-goat antibodies. In summary, we have illustrated how CP can complement different patterning methods to produce repeatedly, and in parallel, high resolution arrays of proteins in three simple steps: 1) TMinking∫, 2) rinsing, and 3) printing the stamp on the substrate. Since -stamps carry the complementary pattern of binding partners specific to the target proteins on their surface, the proteins self-assemble into the predefined array on the stamp surface during inking in solution, and dissociate upon printing. Hence, the (re)production of the target protein arrays is fast and easy. The initial production of the -stamp is a one-time burden only. We thus believe that the methodology presented is powerful and versatile, and should be useful in detection and fabrication strategies that are based on arrays of proteins.

Amplified detection of DNA and analysis of single-base mismatches by the catalyzed deposition of gold on Au-nanoparticles

A novel amplification route for DNA detection based on the deposition of gold on a 10 nm Au-colloid/avidin conjugate label acting as a seeding catalyst, is described. Microgravimetric quartz-crystal-microbalance measurements are employed to transduce the catalyzed deposition of gold on the piezoelectric crystals. Three different DNA detection schemes are described: (i) analysis of a 27-base nucleic acid fragment; (ii) analysis of the entire M13phi DNA (7229 bases); and (iii) detection of a single-base mismatch in a DNA. Ultrasensitive detection of DNA is accomplished by the catalyzed deposition of gold, detection limit approximately 1 x 10(-15) M.

Amplified detection of single-base mismatches in DNA using microgravimetric quartz-crystal-microbalance transduction

Three different methods for the amplified detection of a single-base mismatch in DNA are described using microgravimetric quartz-crystal-microbalance as transduction means. All methods involve the primary incorporation of a biotinylated base complementary to the mutation site in the analyzed double-stranded primer/DNA assembly. The double-stranded assembly is formed between 25 complementary bases of the probe DNA assembled on the Au-quartz crystal and the target DNA. One method of amplification includes the association of avidin- and biotin-labeled liposomes to the sensing interface. The second method of amplified detection of the base mismatch includes the association of an Au-nanoparticle-avidin conjugate to the sensing interface, and the secondary Au-nanoparticle-catalyzed deposition of gold on the particles. The third amplification route includes the binding of the avidin-alkaline phosphatase biocatalytic conjugate to the double-stranded surface followed by the oxidative hydrolysis of 5-bromo-4-chloro-3-indolyl phosphate to the insoluble product indigo derivative that precipitates on the transducer. Comparison of the three amplification routes reveals that the catalytic deposition of gold on the Au-nanoparticle/avidin conjugate is the most sensitive method, and the single-base mismatch in the analyzed DNA is detected with a sensitivity that corresponds to 3x10(-16) M.

Controlled electrocatalysis by microperoxidase-11 and Au-nanoparticle superstructures on conductive supports

Abstract Three-dimensional superstructures, multilayer-arrays consisting of Au-nanoparticles (13±1 nm) and crosslinked by microperoxidase-11 (MP-11), were assembled on transparent ITO conductive glass supports. The structures of the assemblies were confirmed by spectroscopic and electrochemical analyses, which revealed that each Au-particle is associated with ca. 50 MP-11 molecules. The multilayered MP-11/Au-particle systems act as electrocatalysts for the reduction of H2O2. The number of MP-11xa0∣xa0Au-particle layers associated with the electrode controls the resulting electrocatalytic currents. The controllable number of MP-11xa0∣xa0Au-particle layers associated with the electrode enables the control of the effectiveness of the electrocatalytic process and tuning of the sensitivity of the H2O2-sensing interface.

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

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

Biofuel cell based on glucose oxidase and microperoxidase-11 monolayer-functionalized electrodes

Apoglucose oxidase was reconstituted onto a pyrroloquinoline quinone and flavin adenine dinucleotide phosphate, PQQ–FAD, monolayer associated with a rough Au electrode to yield a bioelectrocatalytically active glucose oxidase, GOx. Electrically contacted PQQ–FAD/GOx monolayer was applied to the biocatalytic oxidation of glucose. A microperoxidase-11, MP-11, monolayer was assembled onto a rough Au electrode and used for the biocatalytic reduction of H2O2. Both biocatalytic electrodes, Au/PQQ–FAD/GOx and Au/MP-11, were assembled into a biofuel cell using glucose and H2O2 as the fuel substrate and the oxidizer, respectively. The biofuel cell generates an open-circuit voltage, Voc, of ca. 310 mV and a short-circuit current density, isc, of ca. 114 µA cm–2. The maximum electrical power, Pmax, extracted from the cell is 32 µW at an external optimal load of 3 kΩ. The fill factor of the biofuel cell, fxa0=xa0PmaxIsc–1Voc–1, is ca. 25%.

C60-mediated bioelectrocatalyzed oxidation of glucose with glucose oxidase

Abstract This report emphasizes the use of C 60 as an electron mediator for electrocatalyzed biotransformations. A fullerene, C 60 , carboxylic derivative was covalently attached to a cystamine-monolayer-functionalized Au-electrode. The C 60 -monolayer revealed a quasi-reversible electrochemical behavior ( E 0 =0.31 V vs SCE) in aqueous electrolyte. The C 60 -monolayer (surface density Γ C 60 =1.3×10 −10 mol cm −2 ) provided electrical communication between the electrode and a soluble glucose oxidase, GOx, with the electron transfer rate constant k et =3×10 4 M −1 s −1 . The monolayer-immobilized C 60 -mediated bioelectrocatalyzed glucose oxidation in the presence of GOx which was studied by cyclic voltammetry and the rotating disk electrode technique.

Amplified Telomerase Analysis by Using Rotating Magnetic Particles: The Rapid and Sensitive Detection of Cancer Cells

A highly sensitive telomerase detection method that involves amplified telomerase analysis and the use of rotating magnetic particles has been developed. Magnetic particles, functionalized with a primer (1) that is recognized by telomerase, are mixed with a nucleotide mixture that includes biotinylated‐dUTP, and telomerase‐induced elongation of the primers proceeds with simultaneous biotin incorporation. Avidin–Horseradish peroxidase conjugate, coupled to biotin labels, yields the biocatalytic functional particles. Mixing the resulting particles with naphthoquinone‐modified magnetic particles enables the optoelectronic detection of telomerase. Attraction of the magnetic particles to an electrode, followed by rotation of the particles, causes the electrocatalytic reduction of O2 to H2O2 and HRP‐catalyzed oxidation of luminol (3); this results in chemilumunescence. The intensity of the emitted light depends on the telomerase content of the sample and the rotation speed of the particles. A minimum number of 10 cancer cells could be detected.

Electrical contacting of glucose dehydrogenase by the reconstitution of a pyrroloquinoline quinone-functionalized polyaniline film associated with an Au-electrode: an in situ electrochemical SPR study

A novel method to generate an integrated electrically contacted glucose dehydrogenase electrode by the surface reconstitution of the apo-enzyme on a pyrroloquinoline quinone (PQQ)-modified polyaniline is described. In situ electrochemical surface plasmon resonance (SPR) is used to characterize the bioelectrocatalytic functions of the system.