Manipulating the Folding Landscape of a Multidomain Protein.

Folding of proteins to their functional conformation is paramount to life. Though 75% of the proteome consists of multidomain proteins, our knowledge of folding has been based primarily on studies conducted on single-domain and fast-folding proteins. Nonetheless, the complexity of folding landscapes exhibited by multidomain proteins has received increased scrutiny in recent years. We study the three-domain protein adenylate kinase from E. coli (AK), which has been shown to fold through a series of pathways involving several intermediate states. We use a protein design method to manipulate the folding landscape of AK, and single-molecule FRET spectroscopy to study the effects on the folding process. Mutations introduced in the NMP binding (NMPbind) domain of the protein are found to have unexpected effects on the folding landscape. Thus, while stabilizing mutations in the core of the NMPbind domain retain the main folding pathways of wild-type AK, a destabilizing mutation at the interface between the NMPbind and the CORE domains causes a significant repartition of the flux between the folding pathways. Our results demonstrate the outstanding plasticity of the folding landscape of AK and reveal how specific mutations in the primary structure are translated into changes in folding dynamics. The combination of methodologies introduced in this work should prove useful for deepening our understanding of the folding process of multidomain proteins.

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.

Synthesis of hybrid multicomponent disklike nanoparticles.

This manuscript describes the synthesis of a new generation of multicomponent disklike nanoparticles. In this work, we present for the first time, through the template-based sequential electrochemical deposition of metal/semiconductor/polymer segments, an innovative and effective method for preparing a wide range of metallic, semiconductor, and polymeric hybrid multicomponent disklike nanoparticles covering a wide and controlled dimension range from a few nanometers to hundreds of nanometers. Moreover, we can readily tailor the desired final size, aspect ratio, and composition of the disklike nanoparticles by varying the precursor material used and the electrochemical deposition approach. Furthermore, this simple route leads to a highly reproducible and high-throughput synthetic platform of new multicomponent and multifunctional nanoscale building blocks.