Accelerating PROTAC drug discovery: Establishing a relationship between ubiquitination and target protein degradation.

PROTACs have emerged as a new class of drugs that can target the "undruggable" proteome by hijacking the ubiquitin proteasome system. Despite PROTACs' success, most current PROTACs interface with a limited number of E3 ligases, hindering their expansion to many challenging therapeutic uses. Currently, PROTAC drug discovery relies heavily on traditional Western blotting and reporter gene assays which are insensitive and prone to artifacts, respectively.

Tandem Ubiquitin Binding Entities (TUBEs) as Tools to Explore Ubiquitin-Proteasome System and PROTAC Drug Discovery.

The ubiquitin proteasome system (UPS) is a complex pathway that involves multiple enzymes and culminates in the formation of a polyubiquitin chain on a target protein. As its importance is becoming more evident in drug discovery, there is a renewed interest in understanding the role that polyubiquitin chains play. This has been a challenge, mostly due to the lack of experimental tools for detecting the polyubiquitinated forms of a protein of interest (POI).

An integrated self-powered 3D printed sample concentrator for highly sensitive molecular detection of HIV in whole blood at the point of care.

Rapid and efficient biological sample preparation and pretreatment are crucial for highly sensitive, reliable and reproducible molecular detection of infectious diseases. Herein, we report a self-powered, integrated sample concentrator (SPISC) for rapid plasma separation, pathogen lysis, nucleic acid trapping and enrichment at the point of care. The proposed sample concentrator uses a combination of gravitational sedimentation of blood cells and capillary force for rapid, self-powered plasma separation.

3D-Printed Immunosensor Arrays for Cancer Diagnostics.

Detecting cancer at an early stage of disease progression promises better treatment outcomes and longer lifespans for cancer survivors. Research has been directed towards the development of accessible and highly sensitive cancer diagnostic tools, many of which rely on protein biomarkers and biomarker panels which are overexpressed in body fluids and associated with different types of cancer. Protein biomarker detection for point-of-care (POC) use requires the development of sensitive, noninvasive liquid biopsy cancer diagnostics that overcome the limitations and low sensitivities associated with current dependence upon imaging and invasive biopsies.

Fabrication of Hard-Soft Microfluidic Devices Using Hybrid 3D Printing.

Widely accessible, inexpensive, easy-to-use consumer 3D printers, such as desktop stereolithography (SLA) and fused-deposition modeling (FDM) systems are increasingly employed in prototyping and customizing miniaturized fluidic systems for diagnostics and research. However, these 3D printers are generally limited to printing parts made of only one material type, which limits the functionality of the microfluidic devices without additional assembly and bonding steps. Moreover, mating of different materials requires good sealing in such microfluidic devices.

Real-time Colorimetric Quantitative Molecular Detection of Infectious Diseases on Smartphone-based Diagnostic Platform.

Rapid diagnostics of infectious diseases and accurate identification of their causative pathogens play a crucial role in disease prevention, monitoring, and treatment. Conventional molecular detection of infectious pathogens requires expensive equipment and well-trained personnel, thus limiting its use in centralized clinical laboratories. To address this challenge, a portable smartphone-based quantitative molecular detection platform, termed "smart connected pathogen tracer" (SCPT), has been developed for pathogen monitoring and disease surveillance.

Synergistically enhanced colorimetric molecular detection using smart cup: a case for instrument-free HPV-associated cancer screening.

: Early and accurate detection of disease is crucial for its prevention, identification, and treatment. However, most of disease diagnostics is still limited in clinical laboratories due to the need of complicated instruments and professional personnel. Herein, we reported a smartphone-based synergistically enhanced colorimetric method for molecular diagnostics in our point of care (POC) smart cup platform.

Accessible Telemedicine Diagnostics with ELISA in a 3D Printed Pipette Tip.

We report herein a novel pipet-based "ELISA in a tip" as a new versatile diagnostic tool featuring better sensitivity, shorter incubation time, accessibility, and low sample and reagent volumes compared to traditional ELISA. Capture and analysis of data by a cell phone facilitates electronic delivery of results to health care providers. Pipette tips were designed and 3D printed as adapters to fit most commercial 50-200 μL pipettes.

Automated 4-Sample Protein Immunoassays using 3D-Printed Microfluidics.

Low cost, miniaturized assay platforms that work with small sample volumes, high sensitivity and rapid detection will have high value in future biomolecular diagnostics. Herein we report an automated, 3D printed electrochemiluminescent (ECL) immunoarray integrated with a nanostructured pyrolytic graphite sheet (PGS) microwell chip configured to detect 2 proteins simultaneously from complex liquid samples with high sensitivity and selectivity. Assays are done in 18 min at cost of < $1.

Using Ubiquitin Binders to Decipher the Ubiquitin Code.

Post-translational modifications (PTMs) by ubiquitin (Ub) are versatile, highly dynamic, and involved in nearly all aspects of eukaryote biological function. The reversibility and heterogeneity of Ub chains attached to protein substrates have complicated their isolation, quantification, and characterization. Strategies have emerged to isolate endogenous ubiquitylated targets, including technologies based on the use of Ub-binding peptides, such as tandem-repeated Ub-binding entities (TUBEs).

Automated 3D-Printed Microfluidic Array for Rapid Nanomaterial-Enhanced Detection of Multiple Proteins.

We report here the fabrication and validation of a novel 3D-printed, automated immunoarray to detect multiple proteins with ultralow detection limits. This low cost, miniature immunoarray employs electrochemiluminescent (ECL) detection measured with a CCD camera and employs touch-screen control of a micropump to facilitate automated use. The miniaturized array features prefilled reservoirs to deliver sample and reagents to a paper-thin pyrolytic graphite microwell detection chip to complete sandwich immunoassays.

Pathways of Metabolite-Related Damage to a Synthetic p53 Gene Exon 7 Oligonucleotide Using Magnetic Enzyme Bioreactor Beads and LC-MS/MS Sequencing.

Reactive metabolites of environmental chemicals and drugs can cause site specific damage to the p53 tumor suppressor gene in a major pathway for genotoxicity. We report here a high-throughput, cell-free, 96-well plate magnetic bead-enzyme system interfaced with LC-MS/MS sequencing for bioactivating test chemicals and identifying resulting adduction sites on genes. Bioactivated aflatoxin B1 was reacted with a 32 bp exon 7 fragment of the p53 gene using eight microsomal cytochrome (cyt) P450 enzymes from different organs coated on magnetic beads.

Fully 3D printed integrated reactor array for point-of-care molecular diagnostics.

Molecular diagnostics that involve nucleic acid amplification tests (NAATs) are crucial for prevention and treatment of infectious diseases. In this study, we developed a simple, inexpensive, disposable, fully 3D printed microfluidic reactor array that is capable of carrying out extraction, concentration and isothermal amplification of nucleic acids in variety of body fluids. The method allows rapid molecular diagnostic tests for infectious diseases at point of care.

Ultrathin Graphene-Protein Supercapacitors for Miniaturized Bioelectronics.

Nearly all implantable bioelectronics are powered by bulky batteries which limit device miniaturization and lifespan. Moreover, batteries contain toxic materials and electrolytes that can be dangerous if leakage occurs. Herein, an approach to fabricate implantable protein-based bioelectrochemical capacitors (bECs) employing new nanocomposite heterostructures in which 2D reduced graphene oxide sheets are interlayered with chemically modified mammalian proteins, while utilizing biological fluids as electrolytes is described.

Albumin removal from human serum using surface nanopockets on silica-coated magnetic nanoparticles.

Selective removal of albumin from human serum is an essential step prior to proteomic analyses, especially when using mass spectrometry. Here we report stable synthetic nanopockets on magnetic nanoparticle surfaces that bind to human serum albumin (HSA) with high affinity and specificity. The nanopockets are created by templating HSA on 200 nm silica-coated paramagnetic nanoparticles using polymer layers made using 4 organo-silane monomers.

Automated 3-D Printed Arrays to Evaluate Genotoxic Chemistry: E-Cigarettes and Water Samples.

A novel, automated, low cost, three-dimensional (3-D) printed microfluidic array was developed to detect DNA damage from metabolites of chemicals in environmental samples. The electrochemiluminescent (ECL) detection platform incorporates layer-by-layer (LbL) assembled films of microsomal enzymes, DNA and an ECL-emitting ruthenium metallopolymer in ∼10 nm deep microwells. Liquid samples are introduced into the array, metabolized by the human enzymes, products react with DNA if possible, and DNA damage is detected by ECL with a camera.

Methyl-Cytosine-Driven Structural Changes Enhance Adduction Kinetics of an Exon 7 fragment of the p53 Gene.

Methylation of cytosine (C) at C-phosphate-guanine (CpG) sites enhances reactivity of DNA towards electrophiles. Mutations at CpG sites on the p53 tumor suppressor gene that can result from these adductions are in turn correlated with specific cancers. Here we describe the first restriction-enzyme-assisted LC-MS/MS sequencing study of the influence of methyl cytosines (MeC) on kinetics of p53 gene adduction by model metabolite benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), using methodology applicable to correlate gene damage sites for drug and pollutant metabolites with mutation sites.

3D-printed bioanalytical devices.

While 3D printing technologies first appeared in the 1980s, prohibitive costs, limited materials, and the relatively small number of commercially available printers confined applications mainly to prototyping for manufacturing purposes. As technologies, printer cost, materials, and accessibility continue to improve, 3D printing has found widespread implementation in research and development in many disciplines due to ease-of-use and relatively fast design-to-object workflow. Several 3D printing techniques have been used to prepare devices such as milli- and microfluidic flow cells for analyses of cells and biomolecules as well as interfaces that enable bioanalytical measurements using cellphones.

Electrochemistry-based approaches to low cost, high sensitivity, automated, multiplexed protein immunoassays for cancer diagnostics.

Early detection and reliable diagnostics are keys to effectively design cancer therapies with better prognoses. The simultaneous detection of panels of biomarker proteins holds great promise as a general tool for reliable cancer diagnostics. A major challenge in designing such a panel is to decide upon a coherent group of biomarkers which have higher specificity for a given type of cancer.

CHEMICAL SELECTIVITY OF NUCLEOBASE ADDUCTION RELATIVE TO IN VIVO MUTATION SITES ON EXON 7 FRAGMENT OF P53 TUMOR SUPPRESSOR GENE.

Damage to p53 tumor suppressor gene is found in half of all human cancers. Databases integrating studies of large numbers of tumors and cancer cell cultures show that mutation sites of specific p53 codons are correlated with specific types of cancers. If the most frequently damaged p53 codons in vivo correlate with the most frequent chemical damage sites in vitro, predictions of organ-specific cancer risks might result.

3D-printed supercapacitor-powered electrochemiluminescent protein immunoarray.

Herein we report a low cost, sensitive, supercapacitor-powered electrochemiluminescent (ECL) protein immunoarray fabricated by an inexpensive 3-dimensional (3D) printer. The immunosensor detects three cancer biomarker proteins in serum within 35 min. The 3D-printed device employs hand screen printed carbon sensors with gravity flow for sample/reagent delivery and washing.

3D-Printed Fluidic Devices for Nanoparticle Preparation and Flow-Injection Amperometry Using Integrated Prussian Blue Nanoparticle-Modified Electrodes.

A consumer-grade fused filament fabrication (FFF) 3D printer was used to construct fluidic devices for nanoparticle preparation and electrochemical sensing. Devices were printed using poly(ethylene terephthalate) and featured threaded ports to connect polyetheretherketone (PEEK) tubing via printed fittings prepared from acrylonitrile butadiene styrene (ABS). These devices included channels designed to have 800 μm × 800 μm square cross sections and were semitransparent to allow visualization of the solution-filled channels.

Automated multiplexed ECL Immunoarrays for cancer biomarker proteins.

Point-of-care diagnostics based on multiplexed protein measurements face challenges of simple, automated, low-cost, and high-throughput operation with high sensitivity. Herein, we describe an automated, microprocessor-controlled microfluidic immunoarray for simultaneous multiplexed detection of small protein panels in complex samples. A microfluidic sample/reagent delivery cassette was coupled to a 30-microwell detection array to achieve sensitive detection of four prostate cancer biomarker proteins in serum.

Paper-based electrochemiluminescent screening for genotoxic activity in the environment.

A low cost, microfluidic paper electrochemical device (μPED) was fabricated using screen printing of electrodes and heat transfer of patterned wax paper onto filter paper. The μPED features films of a light-emitting ruthenium metallopolymer, microsomal metabolic enzymes, and DNA to detect potential genotoxic pollutant activity in environmental samples. Unlike conventional analytical methods that detect specific pollutant compounds, the μPED was designed to rapidly measure the presence of genotoxic equivalents in environmental samples with the signal related to benzo[a]pyrene (B[a]P) as a reference standard.