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). TUBEs allow the identification and characterization of Ub chains, and novel substrates for deubiquitylases (DUBs) and Ub ligases (E3s). Here we review their impact on purification, analysis of pan or chain-selective polyubiquitylated proteins and underline the biological relevance of this information. Together with peptide aptamers and other Ub affinity-based approaches, TUBEs will contribute to unraveling the secrets of the Ub code.

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. New reliable methods to monitor true PROTAC function (i.e., ubiquitination and subsequent degradation of targets at physiological expression levels) without external tags are essential to accelerate the PROTAC discovery process and to address many unmet therapeutic areas. In this study, we developed a new high-throughput screening technology using "TUBEs" as ubiquitin-binding entities to monitor PROTAC-mediated poly-ubiquitination of native target proteins with exceptional sensitivity. As a proof of concept, targets including BRD3, Aurora A Kinase, and KRAS were used to demonstrate that ubiquitination kinetics can reliably establish the rank order potencies of PROTAC with variable ligands and linkers. PROTAC-treated cell lysates with the highest levels of endogenous target protein ubiquitination - termed "UbMax" - display excellent correlations with DC50 values obtained from traditional Western blots with the added benefits of being high throughput, providing improved sensitivity, and reducing technical errors.

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. The pathogens (e.g., HIV virus) in separated plasma were directly lysed and pathogen nucleic acid was enriched by an integrated, flow-through FTA® membrane in the concentrator, enabling highly efficient nucleic acid preparation. The FTA® membrane of the SPISC is easy to store and transport at room temperature without need for uninterrupted cold chain, which is crucial for point of care sampling in resource-limited settings. The platform has been successfully applied to detect HIV virus in blood samples. Our experiments show that the sample concentrator can achieve a plasma separation efficiency as high as 95% and a detection sensitivity as low as 10 copies per 200 µL blood (∼100 copies per mL plasma) with variability less than 7%. The sample concentrator described is fully compatible with downstream nucleic acid detection and has great potential for early diagnostics, monitoring and management of infectious diseases at the point of care.

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. Among many endeavors to produce user-friendly, semi-automated, and sensitive protein biomarker sensors, 3D printing is rapidly becoming an important contemporary tool for achieving these goals. Supported by the widely available selection of affordable desktop 3D printers and diverse printing options, 3D printing is becoming a standard tool for developing low-cost immunosensors that can also be used to make final commercial products. In the last few years, 3D printing platforms have been used to produce complex sensor devices with high resolution, tailored towards researchers' and clinicians' needs and limited only by their imagination. Unlike traditional subtractive manufacturing, 3D printing, also known as additive manufacturing, has drastically reduced the time of sensor and sensor array development while offering excellent sensitivity at a fraction of the cost of conventional technologies such as photolithography. In this review, we offer a comprehensive description of 3D printing techniques commonly used to develop immunosensors, arrays, and microfluidic arrays. In addition, recent applications utilizing 3D printing in immunosensors integrated with different signal transduction strategies are described. These applications include electrochemical, chemiluminescent (CL), and electrochemiluminescent (ECL) 3D-printed immunosensors. Finally, we discuss current challenges and limitations associated with available 3D printing technology and future directions of this field.

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. Capture antibodies (Ab1) are immobilized on the inner walls of the pipet tip, which serves as the assay compartment where samples and reagents are moved in and out by pipetting. Signals are generated using colorimetric or chemiluminescent (CL) reagents and can be quantified using a cell phone, CCD camera, or plate reader. We utilized pipet-tip ELISA to detect four cancer biomarker proteins with detection limits similar to or lower than microplate ELISAs at 25% assay cost and time. Recoveries of these proteins from spiked human serum were 85-115% or better, depending slightly on detection mode. Using CCD camera quantification of CL with femto-luminol reagent gave limits of detection (LOD) as low as 0.5 pg/mL. Patient samples (13) were assayed for 3 biomarker proteins with results well correlated to conventional ELISA and an established microfluidic electrochemical immunoassay.

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. All cyt P450s converted aflatoxin B1 to aflatoxin B1-8,9-epoxide that adducts guanine (G) in codon 249, with subsequent depurination to give abasic sites and then strand breaks. This is the first demonstration in a cell-free medium that the aflatoxin B1 metabolite selectively causes abasic site formation and strand breaks at codon 249 of the p53 probe, corresponding to the chemical pathway and mutations of p53 in human liver cells and tumors. Molecular modeling supports the view that binding of aflatoxin B1-8,9-epoxide to G in codon 249 precedes the SN2 adduction reaction. Among a range of metabolic enzymes characteristic of different organs, human liver microsomes and cyt P450 3A5 supersomes showed the highest bioactivation rate for p53 exon 7 damage. This method of identifying metabolite-related gene damage sites may facilitate predictions of organ specific cancers for test chemicals via correlations with mutation sites.

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. The detection chip achieves high sensitivity by using single-wall carbon nanotube-antibody conjugates in the microwells and employing massively labeled antibody-decorated RuBPY-silica nanoparticles to generate ECL. The total cost of an array is $0.65, and an eight-protein assay can be done in duplicate for $0.14 per protein with limits of detection (LOD) as low as 78-110 fg mL-1 in diluted serum. The electronic control system costs $210 in components. Utility of the automated immunoarray was demonstrated by detecting an eight-protein prostate cancer biomarker panel in human serum samples in 25 min. The system is well suited to future clinical and point-of-care diagnostic testing and could be used in resource-limited environments.

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. The second big challenge is to develop test devices to measure these biomarkers quantitatively with high sensitivity and specificity, such that there are no interferences from the complex serum or tissue matrices. Lastly, integrating all these tests into a technology that does not require exclusive training to operate, and can be used at point-of-care (POC) is another potential bottleneck in futuristic cancer diagnostics. In this article, we review electrochemistry-based tools and technologies developed and/or used in our laboratories to construct low-cost microfluidic protein arrays for the highly sensitive detection of a panel of cancer-specific biomarkers with high specificity which at the same time has the potential to be translated into POC applications.

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. This review focuses on preparation and applications of 3D-printed bioanalytical devices.

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. A 3D-printed device with a Y-shaped mixing channel was used to prepare Prussian blue nanoparticles (PBNPs) under flow rates of 100 to 2000 µL min(-1). PBNPs were then attached to gold electrodes for hydrogen peroxide sensing. 3D-printed devices used for electrochemical measurements featured threaded access ports into which a fitting equipped with reference, counter, and PBNP-modified working electrodes could be inserted. PBNP-modified electrodes enabled amperometric detection of H2O2 in the 3D-printed channel by flow-injection analysis, exhibiting a detection limit of 100 nM and linear response up to 20 µM. These experiments show that a consumer-grade FFF printer can be used to fabricate low-cost fluidic devices for applications similar to those that have been reported with more expensive 3D-printing methods.

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. The proteins are prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), platelet factor-4 (PF-4), and interlukin-6 (IL-6). The six channel system is driven by integrated micropumps controlled by an inexpensive programmable microprocessor. The reagent delivery cassette and detection array feature channels made by precision-cut 0.8 mm silicone gaskets. Single-wall carbon nanotube forests were grown in printed microwells on a pyrolytic graphite detection chip and decorated with capture antibodies. The detection chip is housed in a machined microfluidic chamber with a steel metal shim counter electrode and Ag/AgCl reference electrode for electrochemiluminescent (ECL) measurements. The preloaded sample/reagent cassette automatically delivers samples, wash buffers, and ECL RuBPY-silica-antibody detection nanoparticles sequentially. An onboard microcontroller controls micropumps and reagent flow to the detection chamber according to a preset program. Detection employs tripropylamine, a sacrificial reductant, while applying 0.95 V vs Ag/AgCl. Resulting ECL light was measured by a CCD camera. Ultralow detection limits of 10-100 fg mL(-1) were achieved in simultaneous detection of the four protein in 36 min assays. Results for the four proteins in prostate cancer patient serum gave excellent correlation with those from single-protein ELISA.

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. The analytical end point is the detection of DNA damage from metabolites produced in the device using an electrochemiluminescence output measured with a charge-coupled device (CCD) camera. Proof-of-concept of this measurement was established for smoke, water, and food samples. The µPED provides a rapid screening tool for on-site environmental monitoring that specifically monitors the genotoxic reactivity of metabolites of toxic compounds present in the samples.

A microfluidic electrochemiluminescent device for detecting cancer biomarker proteins.

We describe an electrochemiluminescence (ECL) immunoarray incorporated into a prototype microfluidic device for highly sensitive protein detection and apply this system to accurate, sensitive measurements of prostate-specific antigen (PSA) and interleukin-6 (IL-6) in serum. The microfluidic system employed three molded polydimethylsiloxane (PDMS) channels on a conductive pyrolytic graphite chip (2.5 × 2.5 cm) inserted into a machined chamber and interfaced with a pump, switching valve, and sample injector. Each of the three PDMS channels encompasses three 3 µL analytical wells. Capture-antibody-decorated single-wall carbon nanotube forests are fabricated in the bottom of the wells. The antigen is captured by these antibodies on the well bottoms. Then, a RuBPY-silica-secondary antibody (Ab2) label is injected to bind to antigen on the array, followed by injection of sacrificial reductant tripropylamine (TPrA) to produce ECL. For detection, the chip is placed into an open-top ECL measuring cell, and the channels are in contact with electrolyte in the chamber. Potential applied at 0.95 V versus Ag/AgCl oxidizes TPrA to produce ECL by redox cycling the RuBPY species in the particles, and ECL light is measured by a charge-coupled device camera. This approach achieved ultralow detection limits of 100 fg mL(-1) for PSA (9 zeptomole) and 10 fg mL(-1) (1 zeptomole) for IL-6 in calf serum, a 10-25-fold improvement of a similar non-microfluidic array. PSA and IL-6 in synthetic cancer patient serum samples were detected in 1.1 h and results correlated well with single-protein enzyme-linked immunosorbent assays.

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). Tandem Ubiquitin Binding Entities (TUBEs) are engineered protein domains that bind specifically to polyubiquitin chains. These polyubiquitin affinity matrices are highly sensitive as they bind to polyubiquitin chains in the nanomolar range. They exist in two forms: pan-selective TUBEs and chain-selective TUBEs. The ability of TUBEs to be conjugated to different entities is truly what makes them unique. TUBEs are used in a wide variety of experiments such as in protein pulldowns to enrich for polyubiquitinated proteins. They are an alternative to ubiquitin antibodies in Western blots. Further, TUBEs are used as capture reagents for immobilizing polyubiquitinated proteins on a microtiter plate. The use of TUBEs as components of in vitro and cell-based assays presents the unique feature of confirming and assessing the polyubiquitination of a POI in response to inhibitors, activators, or PROTAC® molecules. Therefore, TUBEs not only play a big role in studying the UPS but also have a huge potential for speeding up the drug discovery process.

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. The platform takes advantage of synergistically enhanced colorimetric loop-mediated isothermal amplification (LAMP) assay and smartphone-based color analysis, enabling simple, rapid and reliable nucleic acid quantification without need for expensive fluorescence detection equipment. The SCPT platform has been successfully applied to quantitatively detect: i) HPV DNA in saliva and clinical vaginal swab samples, and ii) HIV RNA in plasma samples with comparable sensitivity to state-of-art machine. It has also been demonstrated for disease spatiotemporal mapping and pathogen tracking by wireless connection and web-based surveillance. Such simple, cost-affordable, portable molecular detection platform has great potential for on-site early disease detection, remote healthcare monitoring, and epidemic surveillance.

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. Here, we report methods to print hybrid structures comprising a hard, rigid component (clear polymethacrylate polymer) printed by a low-cost SLA printer, and where the first printed part is accurately mated and adhered to a second, soft, flexible component (thermoplastic polyurethane elastomer) printed by an FDM printer. The prescribed mounting and alignment of the first-printed SLA-printed hard component, and its pre-treatment and heating during the second FDM step, can produce leak-free bonds at material interfaces. To demonstrate the utility of such hybrid 3D-printing, we prototype and test three components: i) finger-actuated pump, ii) quick-connect fluid coupler, and iii) nucleic acid amplification test device with screw-type twist sealing for sample introduction.

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

Rationale: 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. Methods: A disposable microfluidic chip was developed for colorimetric loop-mediated isothermal amplification (LAMP) detection of multiple HPV DNA in our POC smart cup platform. The colorimetric detection takes advantage of synergistic effect of PPi4- and H+ ions, two byproducts of LAMP reaction. Color signal of LAMP assay was recorded and analyzed by our custom Android app (dubbed "Hue Analyzer"). Results: Our method not only significantly improves colorimetric readout, but also provides a 10-fold increase in detection sensitivity. It has been successfully applied for HPV-associated cancer screening with spiked saliva and clinical swab samples. Conclusion: The proposed POC diagnostic platform is completely compatible with other nucleic acid biomarkers and has great potential for personalized health monitoring and disease prevention.

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 < $1.00 using 1-2 microliters of sample. 3D printed microfluidic array design integrates reagent and sample chambers with rapid ECL detection. A commercial programmable syringe pump used with a preset program allows pump to pause and resume reagent delivery as required for completion of the sandwich immunoassays. Nanostructured surfaces feature antibody-decorated single wall carbon nanotube forests on PGS chip microwells, and sensitivity is amplified via massively labeled RuBPY-silica nanoparticles for detection. Prostate specific antigen (PSA) and prostate specific membrane antigen (PSMA) were measured simultaneously from human serum on the immunoarray with detection limits 150 fg mL-1 for PSA and 230 fg mL-1 for PSMA, with dynamic ranges up to 5 ng mL-1. Validation of the immunoarray by measuring these proteins in human serum showed good correlation with single protein ELISA. These 3D printed platforms can be easily adapted to multiple applications and configurable CAD files for the immunoarray can be downloaded from our lab's website.

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. A simple leak-proof polymerization strategy was developed to integrate flow-through nucleic acid isolation membranes into microfluidic devices, yielding a multifunctional diagnostic platform. Static coating technology was adopted to improve the biocompatibility of our 3D printed device. We demonstrated the suitability of our device for both end-point colorimetric qualitative detection and real-time fluorescence quantitative detection. We applied our diagnostic device to detection of Plasmodium falciparum in plasma samples and Neisseria meningitides in cerebrospinal fluid (CSF) samples by loop-mediated, isothermal amplification (LAMP) within 50 min. The detection limits were 100 fg for P. falciparum and 50 colony-forming unit (CFU) for N. meningitidis per reaction, which are comparable to that of benchtop instruments. This rapid and inexpensive 3D printed device has great potential for point-of-care molecular diagnosis of infectious disease in resource-limited settings.