A silicon chip-based electric detector coupled to bead-based sandwich hybridization (BBSH) is presented as an approach to perform rapid analysis of specific nucleic acids. A microfluidic platform incorporating paramagnetic beads with immobilized capture probes is used for the bio-recognition steps. The protocol involves simultaneous sandwich hybridization of a single-stranded nucleic acid target with the capture probe on the beads and with a detection probe in the reaction solution, followed by enzyme labeling of the detection probe, enzymatic reaction, and finally, potentiometric measurement of the enzyme product at the chip surface. Anti-DIG-alkaline phosphatase conjugate was used for the enzyme labeling of the DIG-labeled detection probe. p-Aminophenol phosphate (pAPP) was used as a substrate. The enzyme reaction product, p-aminophenol (pAP), is oxidized at the anode of the chip to quinoneimine that is reduced back to pAP at the cathode. The cycling oxidation and reduction of these compounds result in a current producing a characteristic signal that can be related to the concentration of the analyte. The performance of the different steps in the assay was characterized using in vitro synthesized RNA oligonucleotides and then the instrument was used for analysis of 16S rRNA in Escherichia coli extract. The assay time depends on the sensitivity required. Artificial RNA target and 16S rRNA, in amounts ranging from 10(11) to 10(10) molecules, were assayed within 25 min and 4 h, respectively.

Kinases are one of the largest families of homologous enzymes encoded by the human genome and are essential regulators of cell communication, immune regulation, stem cell differentiation and many other important metabolic pathways in the body. The identification of kinases, their substrates, and prospective inhibitors is thus critical for understanding various signal transduction cascades and for potential diagnostic and drug discovery applications. [1] Conventional methods for kinase detection typically involve radiolabelling of the substrate using g-32P-ATP or use of a variety of fluorescence-based approaches. [2,3] In this paper, we describe a simple, homogeneous and generic one-step approach for colorimetric kinase detection using functionalized gold nanoparticles (NPs) in solution. The use of inorganic NPs as biosensing platforms is favorable due to their remarkable broad range of optical, chemical, electronic and structural properties which can be used to address some of the limitations of isotopic and fluorescence based assays. [4] The dispersion-dependent absorbance exhibited by suspensions of gold NPs due to interparticle plasmon coupling can be used to provide a convenient optical signal for the detection of protein kinase activity. This general concept has been exploited in several enzyme-responsive gold NP studies. [5,6] For example, kinase-catalyzed biotinylation of peptide-coated gold NPs by means of a g-biotin ATP derivative has been detected using streptavidin-coated gold NPs. The resultant particle aggregates arising from the specific high-affinity streptavidin-biotin interaction leads to a marked color change of the colloid suspension, which can be readily monitored spectroscopically or by eye. [7] The detection format of the assay, however, has the drawback of being a two-stage process. Moreover, there exists no standard method for peptide assembly which can be applied across different colloidal systems without considerably compromising the NP stability. This cumbersome need for case-bycase optimization of the NP assembly thus limits the facile implementation of the technology.The simple and effective assay format we propose comprises a single step and eliminates the need for any labeled ATP derivatives. The system consists of two populations of gold NPs; namely one population coated with a protein kinase substrate peptide and the other coated with complementary antiphosphotyrosine antibodies. Simultaneous addition of enzyme and ATP to the two particle types results in enzymatic phosphorylation of the NP-immobilized peptide substrate and interparticle cross-linking due to specific recognition by the antibody-functionalized particles (Figure 1). This is marked by changes in absorbance intensity at the plasmon resonance peak at 529 nm (typical of stable gold dispersions), and in the visible deposition of NP clusters. Aggregation does not occur in the absence of enzyme or ATP, or in the presence of an inhibitor of either the kinase or the antibody, indicating that this is a specific enzyme-driv...

Peptide microarrays displaying biologically active small synthetic peptides in a high-density format provide an attractive technology to probe complex samples for the presence and/or function of protein analytes. We present a new approach for manufacturing functional peptide microarrays for molecular immune diagnostics. Our method relies on the efficiency of site-specific solution-phase coupling of biotinylated synthetic peptides to NeutrAvidin (NA) and localized microdispensing of peptide-NA-complexes onto activated glass surfaces. Antibodies are captured in a sandwich manner between surface immobilized peptide probes and fluorescence-labeled secondary antibodies. Our work includes a total of 54 peptides derived from immunodominant linear epitopes of the T7 phage capsid protein, Herpes simplex virus glycoprotein D, c-myc protein, and three domains of the Human coronavirus polymerase polyprotein and their cognate mAbs. By using spacer molecules of different type and length for NA-mediated peptide presentation, we show that the incorporation of a minimum spacer length is imperative for antibody binding, whereas the peptide immobilization direction has only secondary importance for antibody affinity and binding. We further demonstrate that the peptide array is capable of detecting low-picomolar concentrations of mAbs in buffered solutions and diluted human serum with high specificity.

A single-step gold nanoparticle (AuNP)-based immunoassay is demonstrated in which the nanoparticle surface is tagged with short viral peptide epitopes. Antiviral antibodies with monoclonal specificity trigger nanoparticle aggregation yielding a colorimetric response that enables detection of antibodies in the low-nanomolar range within a few minutes. In silico insights into the interactions at the epitope−gold interface demonstrate that the conformational landscape exhibited by the epitopes is strongly influenced by the amino acid sequence and location of particular residues within the peptides. The conformation, orientation, and linker chemistry of the peptides affect the immune complex formation in nonintuitive ways that are, nevertheless, explained by a unique sterically kinetically driven aggregation mechanism. The rapid and specific performance of the AuNP immunoassay may have generic potential in point of care diagnostics and other laboratory routines.

We studied the conformational and self-assembly properties of de novo peptides derived from the structure of the yeast transcriptional activator GCN4 in solution and on surfaces. We showed how the pH and the polarity of the solvent directed peptide conformation and assembly. The peptides selfassembled into a coiled coil structure below pH 5 whereas they disassembled above pH 7. The presence of acetonitrile in the peptide solution influenced the peptide's ability to assemble into a coiled coil structure while retaining its alpha-helix conformation. We used these properties to control the peptide packing density on gold surfaces and demonstrated that such peptide-engineered surfaces could be used for reversible molecular binding by utilising functionalised gold nanoparticles.