A Practical Guide to 3D Printing for Chemistry and Biology Laboratories.

Three-dimensional (3D) printing promises a revolution in laboratory creativity by enabling rapid prototyping, broader availability of scientific apparatuses, and transformative scientific workflows. We believe all chemistry and biology laboratories should equip themselves with one or more 3D printers and a critical mass of scientists trained to operate them. This overview surveys the techniques, intricacies, and pitfalls associated with 3D printing of functional parts, including measurements, computer-aided design, slicing, limitations of 3D printing, troubleshooting, tips for tricky filaments, and 3D printer maintenance.

Ultrafast His-Tagged Protein Purification.

This article details how to use a vortex fluidic device (VFD) to accelerate protein purification via immobilized metal affinity chromatography (IMAC). Building upon a previous report of VFD-based purification, we introduce a membrane insert to simplify the purification protocol and the resin recovery step. This new platform can be adapted to different types of IMAC resins and purification membranes.

Catalyst-Free, Three-Component Synthesis of Amidinomaleimides.

Maleimide and amidine functionalities often appear in medicinal and natural product targets. We describe a catalyst-free, three-component coupling reaction for the synthesis of amidinomaleimides. This one-pot reaction fuses a broad range of secondary amines and aldehydes with azidomaleimides.

Printable Hydrogel Arrays for Portable and High-Throughput Shear-Mediated Assays.

Hydrogels have been widely used to entrap biomolecules for various biocatalytic reactions. However, solute diffusion in these matrices to initiate such reactions can be a very slow process. Conventional mixing remains a challenge as it can cause irreversible distortion or fragmentation of the hydrogel itself.

Phage vs. Phage: Direct Selections of Sandwich Binding Pairs.

The sandwich format immunoassay is generally more sensitive and specific than more common assay formats, including direct, indirect, or competitive. A sandwich assay, however, requires two receptors to bind non-competitively to the target analyte. Typically, pairs of antibodies (Abs) or antibody fragments (Fabs) that are capable of forming a sandwiching with the target are identified through a slow, guess-and-check method with panels of candidate binding partners.

Orthogonal glycolytic pathway enables directed evolution of noncanonical cofactor oxidase.

Noncanonical cofactor biomimetics (NCBs) such as nicotinamide mononucleotide (NMN) provide enhanced scalability for biomanufacturing. However, engineering enzymes to accept NCBs is difficult. Here, we establish a growth selection platform to evolve enzymes to utilize NMN-based reducing power.

Evidence for deleterious effects of immunological history in SARS-CoV-2.

A previous report demonstrated the strong association between the presence of antibodies binding to an epitope region from SARS-CoV-2 nucleocapsid, termed Ep9, and COVID-19 disease severity. Patients with anti-Ep9 antibodies (Abs) had hallmarks of antigenic interference (AIN), including early IgG upregulation and cytokine-associated injury. Thus, the immunological memory of a prior infection was hypothesized to drive formation of suboptimal anti-Ep9 Abs in severe COVID-19 infections.

Reengineering the specificity of the highly selective Clostridium botulinum protease via directed evolution.

The botulinum neurotoxin serotype A (BoNT/A) cuts a single peptide bond in SNAP25, an activity used to treat a wide range of diseases. Reengineering the substrate specificity of BoNT/A's protease domain (LC/A) could expand its therapeutic applications; however, LC/A's extended substrate recognition (≈ 60 residues) challenges conventional approaches. We report a directed evolution method for retargeting LC/A and retaining its exquisite specificity.

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.

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.

Single-molecule Taq DNA polymerase dynamics.

Taq DNA polymerase functions at elevated temperatures with fast conformational dynamics-regimes previously inaccessible to mechanistic, single-molecule studies. Here, single-walled carbon nanotube transistors recorded the motions of Taq molecules processing matched or mismatched template-deoxynucleotide triphosphate pairs from 22° to 85°C. By using four enzyme orientations, the whole-enzyme closures of nucleotide incorporations were distinguished from more rapid, 20-μs closures of Taq's fingers domain testing complementarity and orientation.

Synthesis of a High Affinity Complementary Peptide-Polymer Nanoparticle (NP) Pair Using Phage Display.

Peptide-polymer complementary pairs can provide useful tools for isolating, organizing, and separating biomacromolecules. We describe a procedure for selecting a high affinity complementary peptide-polymer nanoparticle (NP) pair using phage display. A hydrogel copolymer nanoparticle containing a statistical distribution of negatively charged and hydrophobic groups was used to select a peptide sequence from a phage displayed library of >10 peptides.

Making the cut with protease engineering.

Proteases cut with enviable precision and regulate diverse molecular events in biology. Such qualities drive a seemingly inexhaustible appetite for proteases with new activities and capabilities. Comprising 25% of the total industrial enzyme market, proteases appear in consumer goods, such as detergents, textile processing, and numerous foods; additionally, proteases include 25 US Food and Drug Administration-approved medicines and various research tools.

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.

Evidence for Deleterious Antigenic Imprinting in SARS-CoV-2 Immune Response.

Unlabelled: A previous report demonstrated the strong association between the presence of antibodies binding to an epitope region from SARS-CoV-2 nucleocapsid, termed Ep9, and COVID-19 disease severity. Patients with anti-Ep9 antibodies (Abs) had hallmarks of antigenic imprinting (AIM), including early IgG upregulation and cytokine-associated injury. Thus, the immunological memory of a previous infection was hypothesized to drive formation of suboptimal anti-Ep9 Abs in severe COVID-19 infections.

Vortex Fluidic-Mediated Fabrication of Fast Gelated Silica Hydrogels with Embedded Laccase Nanoflowers for Real-Time Biosensing under Flow.

The fabrication of hybrid protein-Cu(PO) nanoflowers (NFs) via an intermediate toroidal structure is dramatically accelerated under shear using a vortex fluidic device (VFD), which possesses a rapidly rotating angled tube. As-prepared laccase NFs (LNFs) exhibit ≈1.8-fold increase in catalytic activity compared to free laccase under diffusion control, which is further enhanced by ≈ 2.

Predicting COVID-19 Severity with a Specific Nucleocapsid Antibody plus Disease Risk Factor Score.

Effective methods for predicting COVID-19 disease trajectories are urgently needed. Here, ELISA and coronavirus antigen microarray (COVAM) analysis mapped antibody epitopes in the plasma of COVID-19 patients (n = 86) experiencing a wide-range of disease states. The experiments identified antibodies to a 21-residue epitope from nucleocapsid (termed Ep9) associated with severe disease, including admission to the ICU, requirement for ventilators, or death.

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.

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.

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 Ca.

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.

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.