Open Source, 3D-Printed TrapGuard to Protect Oil-Sealed Vacuum Pumps from Cold Trap Warming.

Clean pump oil is critical to the performance and longevity of oil-sealed vacuum pumps. Cold traps charged with cryogens can protect pump oil from solvent contamination but are subject to operator error. Notably, cold traps with evaporated or warmed cryogens do not protect the vacuum pump. Here, we report an open source device to automatically protect oil-sealed vacuum pumps from cold trap warming and facilitate the daily maintenance of cold traps.

Under-5-Minute Immunoblot Assays by Vortex Fluidic Device Acceleration.

Unlocking the potential of personalized medicine in point-of-care settings requires a new generation of biomarker and proteomic assays. Ideally, assays could inexpensively perform hundreds of quantitative protein measurements in parallel at the bedsides of patients. This goal greatly exceeds current capabilities. Furthermore, biomarker assays are often challenging to translate from benchtop to clinic due to difficulties achieving and assessing the necessary selectivity, sensitivity, and reproducibility. To address these challenges, we developed an efficient (<5 min), robust (comparatively lower CVs), and inexpensive (decreasing reagent use and cost by >70 %) immunoassay method. Specifically, the immunoblot membrane is dotted with the sample and then developed in a vortex fluidic device (VFD) reactor. All assay steps-blocking, binding, and washing-leverage the unique thin-film microfluidics of the VFD. The approach can accelerate direct, indirect, and sandwich immunoblot assays. The applications demonstrated include assays relevant to both the laboratory and the clinic.

Enhancing the Sensitivity of the Virus BioResistor by Overoxidation: Detecting IgG Antibodies.

The Virus BioResistor (VBR) is a biosensor capable of rapid and sensitive detection of small protein disease markers using a simple dip-and-read modality. For example, the bladder cancer-associated protein DJ-1 (22 kDa) can be detected in human urine within 1.0 min with a limit of detection (LOD) of 10 pM. The VBR uses engineered virus particles as receptors to recognize and selectively bind the protein of interest. These virus particles are entrained in a conductive poly(3,4-ethylenedioxythiophene) or PEDOT channel. The electrical impedance of the channel increases when the target protein is bound by the virus particles. But VBRs exhibit a sensitivity that is inversely related to the molecular weight of the protein target. Thus, large proteins, such as IgG antibodies (150 kDa), can be undetectable even at high concentrations. We demonstrate that the electrochemical overoxidation of the VBR's PEDOT channel increases its electrical impedance, conferring enhanced sensitivity for both small and large proteins. Overoxidation makes possible the detection of two antibodies, undetectable at a normal VBR, with a limit of detection of 40 ng/mL (250 pM), and a dynamic range for quantitation extending to 600 ng/mL.

Viruses Masquerading as Antibodies in Biosensors: The Development of the Virus BioResistor.

The 2018 Nobel Prize in Chemistry recognized in vitro evolution, including the development by George Smith and Gregory Winter of phage display, a technology for engineering the functional capabilities of antibodies into viruses. Such bacteriophages solve inherent problems with antibodies, including their high cost, thermal lability, and propensity to aggregate. While phage display accelerated the discovery of peptide and protein motifs for recognition and binding to proteins in a variety of applications, the development of biosensors using intact phage particles was largely unexplored in the early 2000s. Virus particles, 16.5 MDa in size and assembled from thousands of proteins, could not simply be substituted for antibodies in any existing biosensor architectures.Incorporating viruses into biosensors required us to answer several questions: What process will allow the incorporation of viruses into a functional bioaffinity layer? How can the binding of a protein disease marker to a virus particle be electrically transduced to produce a signal? Will the variable salt concentration of a bodily fluid interfere with electrical transduction? A completely new biosensor architecture and a new scheme for electrical transduction of the binding of molecules to viruses were required.This Account describes the highlights of a research program launched in 2006 that answered these questions. These efforts culminated in 2018 in the invention of a biosensor specifically designed to interface with virus particles: the Virus BioResistor (VBR). The VBR is a resistor consisting of a conductive polymer matrix in which M13 virus particles are entrained. The electrical impedance of this resistor, measured across 4 orders of magnitude in frequency, simultaneously measures the concentration of a target protein and the ionic conductivity of the medium in which the resistor is immersed. Large signal amplitudes coupled with the inherent simplicity of the VBR sensor design result in high signal-to-noise ratio (S/N > 100) and excellent sensor-to-sensor reproducibility. Using this new device, we have measured the urinary bladder cancer biomarker nucleic acid deglycase (DJ-1) in urine samples. This optimized VBR is characterized by extremely low sensor-to-sensor coefficients of variation in the range of 3-7% across the DJ-1 binding curve down to a limit of quantitation of 30 pM, encompassing 4 orders of magnitude in concentration.

Photostable and Proteolysis-Resistant Förster Resonance Energy Transfer-Based Calcium Biosensor.

Molecular sensors from protein engineering offer new methods to sensitively bind to and detect target analytes for a wide range of applications. For example, these sensors can be integrated into probes for implantation, and then yield new and valuable physiological information. Here, a new Förster resonance energy transfer (FRET)-based sensor is integrated with an optical fiber to yield a device measuring free Ca2+. This membrane encapsulated optical fiber (MEOF) device is composed of a sensor matrix that fills poly(tetrafluoroethylene) (PTFE) with an engineered troponin C (TnC) protein fused to a pair of FRET fluorophores. The FRET efficiency is modulated upon Ca2+ ion binding. The probe further comprises a second, size-excluding filter membrane that is synthesized by filling the pores of a PTFE matrix with a poly(ethylene glycol) dimethacrylate (PEGDMA) hydrogel; this design ensures protection from circulating proteases and the foreign body response. The two membranes are stacked and placed on a thin, silica optical fiber for optical excitation and detection. Results show the biosensor responds to changes in Ca2+ concentration within minutes with a sensitivity ranging from 0.01 to 10 mM Ca2+, allowing discrimination of hyper- and hypocalcemia. Furthermore, the system reversibly binds Ca2+ to allow continuous monitoring. This work paves the way for the use of engineered structure-switching proteins for continuous optical monitoring in a large number of applications.

Virus Bioresistor (VBR) for Detection of Bladder Cancer Marker DJ-1 in Urine at 10 pM in One Minute.

DJ-1, a 20.7 kDa protein, is overexpressed in people who have bladder cancer (BC). Its elevated concentration in urine allows it to serve as a marker for BC. However, no biosensor for the detection of DJ-1 has been demonstrated. Here, we describe a virus bioresistor (VBR) capable of detecting DJ-1 in urine at a concentration of 10 pM in 1 min. The VBR consists of a pair of millimeter-scale gold electrodes that measure the electrical impedance of an ultrathin (≈ 150-200 nm), two-layer polymeric channel. The top layer of this channel (90-105 nm in thickness) consists of an electrodeposited virus-PEDOT (PEDOT is poly(3,4-ethylenedioxythiophene)) composite containing embedded M13 virus particles that are engineered to recognize and bind to the target protein of interest, DJ-1. The bottom layer consists of spin-coated PEDOT-PSS (poly(styrenesulfonate)). Together, these two layers constitute a current divider. We demonstrate here that reducing the thickness of the bottom PEDOT-PSS layer increases its resistance and concentrates the resistance drop of the channel in the top virus-PEDOT layer, thereby increasing the sensitivity of the VBR and enabling the detection of DJ-1. Large signal amplitudes coupled with the inherent simplicity of the VBR sensor design result in high signal-to-noise (S/N > 100) and excellent sensor-to-sensor reproducibility characterized by coefficients of variation in the range of 3-7% across the DJ-1 binding curve down to a concentration of 30 pM, near the 10 pM limit of detection (LOD), encompassing four orders of magnitude in concentration.

Pyrocinchonimides Conjugate to Amine Groups on Proteins via Imide Transfer.

Advances in bioconjugation, the ability to link biomolecules to each other, small molecules, surfaces, and more, can spur the development of advanced materials and therapeutics. We have discovered that pyrocinchonimide, the dimethylated analogue of maleimide, undergoes a surprising transformation with biomolecules. The reaction targets amines and involves an imide transfer, which has not been previously reported for bioconjugation purposes. Despite their similarity to maleimides, pyrocinchonimides do not react with free thiols. Though both lysine residues and the N-termini of proteins can receive the transferred imide, the reaction also exhibits a marked preference for certain amines that cannot solely be ascribed to solvent accessibility. This property is peculiar among amine-targeting reactions and can reduce combinatorial diversity when many available reactive amines are available, such as in the formation of antibody-drug conjugates. Unlike amides, the modification undergoes very slow reversion under high pH conditions. The reaction offers a thermodynamically controlled route to single or multiple modifications of proteins for a wide range of applications.

3D-Printed Labware for High-Throughput Immobilization of Enzymes.

In continuous flow biocatalysis, chemical transformations can occur under milder, greener, more scalable, and safer conditions than conventional organic synthesis. However, the method typically involves extensive screening to optimize each enzyme's immobilization on its solid support material. The task of weighing solids for large numbers of experiments poses a bottleneck for screening enzyme immobilization conditions. For example, screening conditions often require multiple replicates exploring different support chemistries, buffer compositions, and temperatures. Thus, we report 3D-printed labware designed to measure and handle solids in multichannel format and expedite screening of enzyme immobilization conditions. To demonstrate the generality of these advances, alkaline phosphatase, glucose dehydrogenase, and laccase were screened for immobilization efficiency on seven resins. The results illustrate the requirements for optimization of each enzyme's loading and resin choice for optimal catalytic performance. Here, 3D-printed labware can decrease the requirements for an experimentalist's time by >95%. The approach to rapid optimization of enzyme immobilization is applicable to any enzyme and many solid support resins. Furthermore, the reported devices deliver precise and accurate aliquots of essentially any granular solid material.

Continuous flow biocatalysis.

The continuous flow synthesis of active pharmaceutical ingredients, value-added chemicals, and materials has grown tremendously over the past ten years. This revolution in chemical manufacturing has resulted from innovations in both new methodology and technology. This field, however, has been predominantly focused on synthetic organic chemistry, and the use of biocatalysts in continuous flow systems is only now becoming popular. Although immobilized enzymes and whole cells in batch systems are common, their continuous flow counterparts have grown rapidly over the past two years. With continuous flow systems offering improved mixing, mass transfer, thermal control, pressurized processing, decreased variation, automation, process analytical technology, and in-line purification, the combination of biocatalysis and flow chemistry opens powerful new process windows. This Review explores continuous flow biocatalysts with emphasis on new technology, enzymes, whole cells, co-factor recycling, and immobilization methods for the synthesis of pharmaceuticals, value-added chemicals, and materials.

The Virus Bioresistor: Wiring Virus Particles for the Direct, Label-Free Detection of Target Proteins.

The virus bioresistor (VBR) is a chemiresistor that directly transfers information from virus particles to an electrical circuit. Specifically, the VBR enables the label-free detection of a target protein that is recognized and bound by filamentous M13 virus particles, each with dimensions of 6 nm ( w) × 1 µm ( l), entrained in an ultrathin (∼250 nm) composite virus-polymer resistor. Signal produced by the specific binding of virus to target molecules is monitored using the electrical impedance of the VBR: The VBR presents a complex impedance that is modeled by an equivalent circuit containing just three circuit elements: a solution resistance ( Rsoln), a channel resistance ( RVBR), and an interfacial capacitance ( CVBR). The value of RVBR, measured across 5 orders of magnitude in frequency, is increased by the specific recognition and binding of a target protein to the virus particles in the resistor, producing a signal Δ RVBR. The VBR concept is demonstrated using a model system in which human serum albumin (HSA, 66 kDa) is detected in a phosphate buffer solution. The VBR cleanly discriminates between a change in the electrical resistance of the buffer, measured by Rsoln, and selective binding of HSA to virus particles, measured by RVBR. The Δ RVBR induced by HSA binding is as high as 200 Ω, contributing to low sensor-to-sensor coefficients-of-variation (<15%) across the entire calibration curve for HSA from 7.5 nM to 900 nM. The response time for the VBR is 3-30 s.

Directed evolution and biophysical characterization of a full-length, soluble, human caveolin-1 variant.

Protein engineering by directed evolution can alter proteins' structures, properties, and functions. However, membrane proteins, despite their importance to living organisms, remain relatively unexplored as targets for protein engineering and directed evolution. This gap in capabilities likely results from the tendency of membrane proteins to aggregate and fail to overexpress in bacteria cells. For example, the membrane protein caveolin-1 has been implicated in many cell signaling pathways and diseases, yet the full-length protein is too aggregation-prone for detailed mutagenesis, directed evolution, and biophysical characterization. Using a phage-displayed library of full-length caveolin-1 variants, directed evolution with alternating subtractive and functional selections isolated a full-length, soluble variant, termed cavsol, for expression in E. coli. Cavsol folds correctly and binds to its known protein ligands HIV gp41, the catalytic domain of cAMP-dependent protein kinase A, and the polymerase I and transcript release factor. As expected, cavsol does not bind off-target proteins. Cellular studies show that cavsol retains the parent protein's ability to localize at the cellular membrane. Unlike truncated versions of caveolin, cavsol forms large, oligomeric complexes consisting of approximately >50 monomeric units without requiring additional cellular components. Cavsol's secondary structure is a mixture of α-helices and ß-strands. Isothermal titration calorimetry experiments reveal that cavsol binds to gp41 and PKA with low micromolar binding affinity (KD). In addition to the insights into caveolin structure and function, the approach applied here could be generalized to other membrane proteins.

Synthesis and Explosion Hazards of 4-Azido-l-phenylalanine.

A reliable, scalable, cost-effective, and chromatography-free synthesis of 4-azido-l-phenylalanine beginning from l-phenylalanine is described. Investigations into the safety of the synthesis reveal that the Ullman-like Cu(I)-catalyzed azidation step does not represent a significant risk. The isolated 4-azido-l-phenylalanine product, however, exhibits previously undocumented explosive characteristics.

Virus-Enabled Biosensor for Human Serum Albumin.

The label-free detection of human serum albumin (HSA) in aqueous buffer is demonstrated using a simple, monolithic, two-electrode electrochemical biosensor. In this device, both millimeter-scale electrodes are coated with a thin layer of a composite containing M13 virus particles and the electronically conductive polymer poly(3,4-ethylenedioxy thiophene) or PEDOT. These virus particles, engineered to selectively bind HSA, serve as receptors in this biosensor. The resistance component of the electrical impedance, Zre, measured between these two electrodes provides electrical transduction of HSA binding to the virus-PEDOT film. The analysis of sample volumes as small as 50 µL is made possible using a microfluidic cell. Upon exposure to HSA, virus-PEDOT films show a prompt increase in Zre within 5 s and a stable Zre signal within 15 min. HSA concentrations in the range from 100 nM to 5 µM are detectable. Sensor-to-sensor reproducibility of the HSA measurement is characterized by a coefficient-of-variance (COV) ranging from 2% to 8% across this entire concentration range. In addition, virus-PEDOT sensors successfully detected HSA in synthetic urine solutions.

Vortex Fluidic Chemical Transformations.

Driving chemical transformations in dynamic thin films represents a rapidly thriving and diversifying research area. Dynamic thin films provide a number of benefits including large surface areas, high shearing rates, rapid heat and mass transfer, micromixing and fluidic pressure waves. Combinations of these effects provide an avant-garde style of conducting chemical reactions with surprising and unusual outcomes. The vortex fluidic device (VFD) has proved its capabilities in accelerating and increasing the efficiencies of numerous organic, materials and biochemical reactions. This Minireview surveys transformations that have benefited from VFD-mediated processing, and identifies concepts driving the effectiveness of vortex-based dynamic thin films.

Ten-Minute Protein Purification and Surface Tethering for Continuous-Flow Biocatalysis.

Nature applies enzymatic assembly lines to synthesize bioactive compounds. Inspired by such capabilities, we have developed a facile method for spatially segregating attached enzymes in a continuous-flow, vortex fluidic device (VFD). Fused Hisn -tags at the protein termini allow rapid bioconjugation and consequent purification through complexation with immobilized metal affinity chromatography (IMAC) resin. Six proteins were purified from complex cell lysates to average homogeneities of 76 %. The most challenging to purify, tobacco epi-aristolochene synthase, was purified in only ten minutes from cell lysate to near homogeneity (>90 %). Furthermore, this "reaction-ready" system demonstrated excellent stability during five days of continuous-flow processing. Towards multi-step transformations in continuous flow, proteins were arrayed as ordered zones on the reactor surface allowing segregation of catalysts. Ordering enzymes into zones opens up new opportunities for continuous-flow biosynthesis.

Harnessing Thin-Film Continuous-Flow Assembly Lines.

Inspired by nature's ability to construct complex molecules through sequential synthetic transformations, an assembly line synthesis of α-aminophosphonates has been developed. In this approach, simple starting materials are continuously fed through a thin-film reactor where the intermediates accrue molecular complexity as they progress through the flow system. Flow chemistry allows rapid multistep transformations to occur via reaction compartmentalization, an approach not amenable to using conventional flasks. Thin film processing can also access facile in situ solvent exchange to drive reaction efficiency, and through this method, α-aminophosphonate synthesis requires only 443 s residence time to produce 3.22 g h(-1) . Assembly-line synthesis allows unprecedented reaction flexibility and processing efficiency.

Accelerating Enzymatic Catalysis Using Vortex Fluidics.

Enzymes catalyze chemical transformations with outstanding stereo- and regio-specificities, but many enzymes are limited by their long reaction times. A general method to accelerate enzymes using pressure waves contained within thin films is described. Each enzyme responds best to specific frequencies of pressure waves, and an acceleration landscape for each protein is reported. A vortex fluidic device introduces pressure waves that drive increased rate constants (kcat ) and enzymatic efficiency (kcat /Km ). Four enzymes displayed an average seven-fold acceleration, with deoxyribose-5-phosphate aldolase (DERA) achieving an average 15-fold enhancement using this approach. In solving a common problem in enzyme catalysis, a powerful, generalizable tool for enzyme acceleration has been uncovered. This research provides new insights into previously uncontrolled factors affecting enzyme function.

Glutamate provides a key structural contact between reticulon-4 (Nogo-66) and phosphocholine.

Human reticulon 4 (RTN-4) has been identified as the neurite outgrowth inhibitor (Nogo). This protein contains a span of 66 amino acids (Nogo-66) flanked by two membrane helices at the C-terminus. We previously determined the NMR structure of Nogo-66 in a native-like environment and defined the regions of Nogo-66 expected to be membrane embedded. We hypothesize that aromatic groups and a negative charge hyperconserved among RTNs (Glu26) drive the remarkably strong association of Nogo-66 with a phosphocholine surface. Glu26 is an isolated charge with no counterion provided by nearby protein groups. We modeled the docking of dodecylphosphocholine (DPC) with Nogo-66 and found that a lipid choline group could form a stable salt bridge with Glu26 and serve as a membrane anchor point. To test the role of the Glu26 anion in binding choline, we mutated this residue to alanine and assessed the structural consequences, the association with lipid and the affinity for the Nogo receptor. In an aqueous environment, Nogo-66 Glu26Ala is more helical than WT and binds the Nogo receptor with higher affinity. Thus, we can conclude that in the absence of a neutralizing positive charge provided by lipid, the glutamate anion is destabilizing to the Nogo-66 fold. Although the Nogo-66 Glu26Ala free energy of transfer from water into lipid is similar to that of WT, NMR data reveal a dramatic loss of tertiary structure for the mutant in DPC micelles. These data show that Glu26 has a key role in defining the structure of Nogo-66 on a phosphocholine surface. This article is part of a special issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

Processive Incorporation of Deoxynucleoside Triphosphate Analogs by Single-Molecule DNA Polymerase I (Klenow Fragment) Nanocircuits.

DNA polymerases exhibit a surprising tolerance for analogs of deoxyribonucleoside triphosphates (dNTPs), despite the enzymes' highly evolved mechanisms for the specific recognition and discrimination of native dNTPs. Here, individual DNA polymerase I Klenow fragment (KF) molecules were tethered to a single-walled carbon nanotube field-effect transistor (SWCNT-FET) to investigate accommodation of dNTP analogs with single-molecule resolution. Each base incorporation accompanied a change in current with its duration defined by τclosed. Under Vmax conditions, the average time of τclosed was similar for all analog and native dNTPs (0.2 to 0.4 ms), indicating no kinetic impact on this step due to analog structure. Accordingly, the average rates of dNTP analog incorporation were largely determined by durations with no change in current defined by τopen, which includes molecular recognition of the incoming dNTP. All α-thio-dNTPs were incorporated more slowly, at 40 to 65% of the rate for the corresponding native dNTPs. During polymerization with 6-Cl-2APTP, 2-thio-dTTP, or 2-thio-dCTP, the nanocircuit uncovered an alternative conformation represented by positive current excursions that does not occur with native dNTPs. A model consistent with these results invokes rotations by the enzyme's O-helix; this motion can test the stability of nascent base pairs using nonhydrophilic interactions and is allosterically coupled to charged residues near the site of SWCNT attachment. This model with two opposing O-helix motions differs from the previous report in which all current excursions were solely attributed to global enzyme closure and covalent-bond formation. The results suggest the enzyme applies a dynamic stability-checking mechanism for each nascent base pair.