Novel sensor-integrated proteome on chip (SPOC) platform with thousands of folded proteins on a 1.5 sq-cm biosensor chip to enable high-throughput real-time label-free screening for kinetic analysis.

An automated proteomic platform for producing and screening an array of functional proteins on biosensor surfaces was developed to address the challenges of measuring proteomic interaction kinetics in high throughput (HTP). This technology is termed Sensor-Integrated Proteome On Chip (SPOC®) which involves in-situ cell-free protein expression in nano-liter volume wells (nanowells) directly from rapidly customizable arrays of plasmid DNA, facilitating simultaneous capture-purification of up to 2400 unique full-length folded proteins onto a 1.5 sq-cm surface of a single gold biosensor chip. Arrayed SPOC sensors can then be screened by real-time label-free analysis, including surface plasmon resonance (SPR) to generate kinetic affinity, avidity data. Fluorescent and SPR assays were used to demonstrate zero crosstalk between protein spots. The functionality of the SPOC protein array was validated by antibody binding assay, post-translational modification, mutation-mediated differential binding kinetics, and catalytic activity screening on model SPOC protein arrays containing p53, Src, Jun, Fos, HIST1H3A, and SARS-CoV-2 receptor binding domain (RBD) protein variants of interest, among others. Monoclonal antibodies were found to selectively bind their target proteins on the SPOC array. A commercial anti-RBD antibody was used to demonstrate discriminatory binding to numerous SARS-CoV-2 RBD variants of concern with comprehensive kinetic information. With advantages of HTP, flexibility, low-cost, quick turnaround time, and real-time kinetic affinity profiling, the SPOC proteomic platform addresses the challenges of interrogating protein interactions at scale and can be deployed in various research and clinical applications.

A Contra Capture Protein Array Platform for Studying Post-translationally Modified (PTM) Auto-antigenomes.

Aberrant modifications of proteins occur during disease development and elicit disease-specific antibody responses. We have developed a protein array platform that enables the modification of many proteins in parallel and assesses their immunogenicity without the need to express, purify, and modify proteins individually. We used anticitrullinated protein antibodies (ACPAs) in rheumatoid arthritis (RA) as a model modification and profiled antibody responses to ∼190 citrullinated proteins in 20 RA patients. We observed unique antibody reactivity patterns in both clinical anticyclic citrullinated peptide assay positive (CCP+) and CCP- RA patients. At individual antigen levels, we detected antibodies against known citrullinated autoantigens and discovered and validated five novel antibodies against specific citrullinated antigens (osteopontin (SPP1), flap endonuclease (FEN1), insulin like growth factor binding protein 6 (IGFBP6), insulin like growth factor I (IGF1) and stanniocalcin-2 (STC2)) in RA patients. We also demonstrated the utility of our innovative array platform in the identification of immune-dominant epitope(s) for citrullinated antigens. We believe our platform will promote the study of post-translationally modified antigens at a breadth that has not been achieved before, by both identifying novel autoantigens and investigating their roles in disease development. The developed platforms can potentially be used to study many autoimmune disease-relevant modifications and their immunogenicity.

High density diffusion-free nanowell arrays.

Proteomics aspires to elucidate the functions of all proteins. Protein microarrays provide an important step by enabling high-throughput studies of displayed proteins. However, many functional assays of proteins include untethered intermediates or products, which could frustrate the use of planar arrays at very high densities because of diffusion to neighboring features. The nucleic acid programmable protein array (NAPPA) is a robust in situ synthesis method for producing functional proteins just-in-time, which includes steps with diffusible intermediates. We determined that diffusion of expressed proteins led to cross-binding at neighboring spots at very high densities with reduced interspot spacing. To address this limitation, we have developed an innovative platform using photolithographically etched discrete silicon nanowells and used NAPPA as a test case. This arrested protein diffusion and cross-binding. We present confined high density protein expression and display, as well as functional protein-protein interactions, in 8000 nanowell arrays. This is the highest density of individual proteins in nanovessels demonstrated on a single slide. We further present proof of principle results on ultrahigh density protein arrays capable of up to 24000 nanowells on a single slide.

A nanocontact printing system for sub-100 nm aligned patterning.

Though many aspects of contact printing have been explored extensively since its invention, there are still hurdles to overcome for multilayer printing in the nanometer regime. Here we report on an aligned nanocontact printing (nCP) system that has demonstrated a sub-100 nm alignment capability by means of moiré fringes and microspacers. To address issues in the stamp inking, we have devised a microfluidic apparatus based on the gradient capillary force for transport of ink solutions. The nCP system has been tested by printing nucleoside phosphoramidites on a nanopillar arrayed substrate. Although the nCP system was designed primarily for use in the fabrication of high density DNA nanoarrays, it has the potential to be applied to other fields of nanotechnology for nanoscale patterning.

Molecular sensing using monolayer floating gate, fully depleted SOI MOSFET acting as an exponential transducer.

Field-effect transistor-based chemical sensors fall into two broad categories based on the principle of signal transduction-chemiresistor or Schottky-type devices and MOSFET or inversion-type devices. In this paper, we report a new inversion-type device concept-fully depleted exponentially coupled (FDEC) sensor, using molecular monolayer floating gate fully depleted silicon on insulator (SOI) MOSFET. Molecular binding at the chemical-sensitive surface lowers the threshold voltage of the device inversion channel due to a unique capacitive charge-coupling mechanism involving interface defect states, causing an exponential increase in the inversion channel current. This response of the device is in opposite direction when compared to typical MOSFET-type sensors, wherein inversion current decreases in a conventional n-channel sensor device upon addition of negative charge to the chemical-sensitive device surface. The new sensor architecture enables ultrahigh sensitivity along with extraordinary selectivity. We propose the new sensor concept with the aid of analytical equations and present results from our experiments in liquid phase and gas phase to demonstrate the new principle of signal transduction. We present data from numerical simulations to further support our theory.

Electrical detection of amine ligation to a metalloporphyrin via a hybrid SOI-MOSFET.

A close-packed monolayer of zinc 5,10,15,20-tetrakis(3-carboxyphenyl)porphyrin has been prepared and deposited on the thin native oxide covering the surface of an SOI-MOSFET (silicon-on-insulator metal-oxide-semiconductor field effect transistor) using Langmuir-Blodgett techniques. When the device is exposed to amine vapors in a nitrogen atmosphere, the amine coordinates to the zinc atom. The resulting change in electron distribution within the porphyrin leads to a large change in the drain current of the transistor, biased via a back gate. This change is sensitive to both the amount of amine present and the base strength of the amine. Only very small changes in drain current were observed with a monolayer of free base porphyrin or palmitic acid. After exposure to high pyridine concentrations, the device response saturates, but partially recovers after overnight exposure to flowing nitrogen gas. Interestingly, the device response is instantaneously reset by exposure to visible light, suggesting that photode-ligation occurs. An electrical model for the hybrid device that describes its response to ligand binding in terms of a change in the work function of the porphyrin monolayer has been developed. A transistor response to a few hundred attomoles of bound pyridine can be readily detected. This extreme sensitivity, coupled with the ability to reset the device using light, suggests that such systems might be useful as sensors.

A New Approach to Fabricating High-density Nanoarrays by Nanocontact Printing.

We introduce a new scheme of nanocontact printing that fabricates nanoarrays using stamps generated by ultraviolet nanoimprint lithography. Array patterns can be generated by this printing technique in a high-density (number of features per unit area) fashion with a feature size as low as 30 nm and period of 100 nm. Sub-500 nm alignment accuracy for multilayer printing has been obtained using a traditional contact mask aligner. We also demonstrate that we can image a nanoarray labeled by streptavidin by atomic force microscope (AFM).