Zhilei Chen

Urinary tract infections (UTIs) are among the most common bacterial infections, and recurrent UTIs caused by uropathogenic Escherichia coli (UPEC) drive antibiotic use and antimicrobial resistance. FimH, the mannose-binding adhesin at the tip of type 1 pili, is a central UPEC virulence factor. Under shear force, FimH switches from a low-affinity, tense state to a high-affinity, relaxed state, strengthening adhesion during urine flow. This catch-bond behavior makes FimH a promising anti-virulence target, though a difficult one to drug. Here, we review strategies to target FimH, including monovalent and multivalent mannosides, vaccines, conformation-selective monoclonal antibodies, and de novo engineered miniproteins, together with the assays used to quantify FimH-ligand binding and UPEC adhesion. Clinical translation has been limited by assay-dependent potency, urinary pharmacokinetics, and intracellular reservoirs. We posit that effective FimH-targeted therapy requires alignment among inhibitor mechanism, FimH conformational state, bladder lumen exposure, and infection stage, which in turn defines realistic roles in prevention, early intervention, and combination therapy.

DISCO-LAMP combines proximity ligation assay (PLA) with isothermal DNA amplification (LAMP) for protein detection. It sensitively detected Shiga toxin 2 and coronavirus spike proteins, supporting broad disease-target detection.

Shiga toxin-producing Escherichia coli (STEC) are major foodborne pathogens responsible for severe infections, sometimes fatal. However, the current diagnostic methods lack the sensitivity and speed required for effective clinical assessment and food safety inspection. Highly sensitive detection of Shiga toxin 2 (Stx2), a primary virulence factor of STEC, could potentially offer critical benefits for timely intervention. In this work, gold nanoparticles (AuNPs) are functionalized with a pair of high-affinity designed ankyrin repeat proteins (DARPins), targeting the A and B subunits of Stx2, and used as multifunctional signal transducers for rapid electronic detection (RED) of Stx2. This DARPin-RED platform leverages active centrifugal forces, vortex agitation, and a digital readout device for rapid (<30 min), yet highly sensitive (fg/mL-level) detection of Stx2 in food matrices (e.g. milk, lettuce extract, and ground beef extract) and biological fluids. For example, the analytical limits of detection (LoDs) are 14.8, 38, and 25 fg/mL in serum, whole blood, and stool, respectively, supporting future evaluation in well-characterized clinical specimens. Additionally, DARPin-RED can detect multiple Stx2 subtypes with minimal background interference, distinguish different bacterial inoculation doses (p = 0.011), resolve incubation-time-dependent toxin production, and differentiate high-toxin-producing E. coli strains (e.g. RM5856) from low-toxin producers (e.g. RM9872) (p = 0.037). These results establish DARPin-RED as a rapid and sensitive analytical platform for further development in STEC testing and food-safety surveillance.

The cell line and scalable upstream/downstream process development needed for every new monoclonal antibody are both highly expensive and time‐consuming, significantly increasing the cost of new antibody therapeutics. By contrast, the production of many synthetic binders can be accomplished rapidly and with lower production costs. Unfortunately, the clinical efficacy of these synthetic binders is hampered by the lack of antibody Fc effector functions. We report a synthetic modular platform, Bridge Protein (BP), capable of permanently repurposing an FDA‐approved antibody to new therapeutic targets. BP is a bispecific designed ankyrin repeat protein (DARPin) that binds an antibody paratope and a new target of interest. As proof of concept, we created a BP for redirecting ofatumumab, an anti‐CD20 antibody, to HER2 ⁺ cells and demonstrated its ability to induce antibody‐dependent cellular cytotoxicity (ADCC). Incorporation of p ‐benzoyl‐L‐phenylalanine (BPA) in BP enables its covalent photocrosslinking to ofatumumab without compromising target binding or ADCC. This plug‐and‐play platform enables straightforward and cost‐effective antibody retargeting and opens the door to rapidly repurposing stockpiled antibodies for pandemic preparedness and biotherapeutic development.

Proximity ligation assay (PLA), in which the ligation of two DNA probes is greatly accelerated by the associating target molecules, has emerged as a highly sensitive technique for protein detection. The detection of the ligated DNA typically relies on PCR, which requires temperature cycling. In this study, we report on a novel disco ntinuous (DISCO)-LAMP assay that enables the wash-free detection of PLA products via loop-mediated isothermal amplification (LAMP). Due to the exponential amplification nature of LAMP, a careful balance between efficient amplification of the ligated full-length DNA and minimal background amplification from the individual constituent probes is essential but often challenging to achieve. After extensive template/primer design and assay optimization, DISCO-LAMP assay achieved a detection limit of 1 fM for the ligated DNA probe while maintaining undetectable background amplification at 1 nM of each individual probe. DISCO-LAMP detected Shiga toxin 2 (Stx2) with a limit of detection (LoD) of 100 fM when functionalized with Stx2-binders, as well as both Wuhan-1 and Omicron spike protein when functionalized with D _S16 , a newly engineered DARPin targeting a conserved epitope on the SARS-CoV-2 Spike protein. We believe DISCO-LAMP represents a versatile and efficient LAMP-based PLA technology that is readily adaptable for sensing diverse targets.

Rapid and scalable protein binder discovery platforms are essential for accelerating therapeutic and diagnostic development. Click display is a cell-free protein display technology that covalently links nascent proteins to their encoding cDNA in a one-pot reaction within 2 h, enabling rapid in vitro selection. Although previously demonstrated to support directed evolution, enrichment efficiency during biopanning was slower than conventional phage display. Here, we report two modifications that significantly enhance click display selection efficiency: (i) selective elution of target-bound complexes using a cleavable biotin linker, and (ii) competitive displacement of non-specific bead binders using a biotinylated blocking protein. These improvements dramatically reduce amplification of non-specific binders and enable efficient enrichment of target-specific binders within three to four rounds of panning. Using a naïve DARPin library, we rapidly enriched binders to interleukin-10 (IL-10) and transforming growth factor-β1 (TGF-β1), identifying clones with low nanomolar binding affinities and receptor-blocking activity. We further applied the optimized protocol to affinity maturation of a PD-L1-binding DARPin, achieving >400-fold improved binding affinity. The best candidate, 1D10, exhibits sub-nanomolar PD-1 inhibition in competition ELISA. These results demonstrate that the optimized click display workflow enables rapid enrichment and affinity maturation of high-affinity DARPins across diverse targets. The platform provides a streamlined, transformation-free approach for accelerating protein engineering and directed evolution.

Shiga toxin-producing Escherichia coli (STEC) is a major foodborne pathogen, responsible for severe gastrointestinal disease and hemolytic uremic syndrome (HUS). Here, we report Click Detect, a novel diagnostic platform that leverages click display to efficiently produce sensing probes for sandwich-style antigen detection. Click display is an in vitro protein display technology that generates uniform and covalently linked protein–cDNA conjugates in a simple one-pot reaction format within 2 h. The captured sensing probe can be quantified by standard nucleic acid amplification assays. Using click displayed DARPin (D_#20) as the sensing probe and a high-affinity nanobody (N_G1) as the capture reagent, Click Detect reliably detected Shiga toxin 2 (Stx2) at 600 fM by quantitative PCR (qPCR) and 6 pM by loop-mediated isothermal amplification (LAMP). The assay maintained comparable sensitivity in matrices containing up to 40% public swimming pool water or lettuce extract, highlighting robustness for real-world surveillance applications. Key advantages of Click Detect include simple, rapid, and cost-effective (~USD 0.04 per assay) sensing probe preparation, as well as a versatile plug-and-play probe format for detecting other targets. We believe that Click Detect has great potential as a novel sensing platform for food/environmental monitoring and point-of-care diagnostics, with potentially broad applicability to other toxins and protein targets.

Shiga toxin–producing Escherichia coli (STEC) are major foodborne pathogens responsible for severe gastrointestinal disease and hemolytic uremic syndrome (HUS). Here we report Click Detect, a novel diagnostic platform that leverages click display – an in vitro protein display technology – to generate uniform and covalently linked protein–cDNA conjugates for toxin detection. Using a DARPin (D_#20) as the signal probe and a high-affinity nanobody (N_G1) as the capture reagent, Click Detect achieves sandwich-style recognition of Shiga toxin 2 (Stx2). The captured protein–cDNA conjugates can be quantified by standard nucleic acid amplification assays. Click Detect reliably detected Stx2 at 600 fM by qPCR and 6 pM by LAMP. The assay maintained comparable sensitivity in matrices containing up to 40% public swimming pool water or lettuce extract, highlighting robustness in real-world contamination scenarios. Key advantages of Click Detect include simple, rapid, and cost-effective (~$0.04 per assay) probe preparation as well as versatile plug-and-play probe format for sensing other targets. We believe Click Detect has great potential as a novel sensing platform for food safety monitoring, environmental surveillance, and point-of-care diagnostics, with potentially broad applicability to other toxins and protein targets.

Shiga toxin-producing Escherichia coli (STEC) are major foodborne pathogens responsible for severe infections, including the deadly hemolytic uremic syndrome (HUS). However, the current diagnostic methods lack the sensitivity and speed required for effective clinical and food safety applications. Early detection of Shiga toxin 2 (Stx2), a primary virulence factor of STEC, could potentially offer critical benefits for timely intervention. In this work, gold n anoparticles (AuNPs) are functionalized with a pair of high-affinity, d esigned a nkyrin repeat p roteins (DARPins) targeting the A and B subunits of Stx2, and used as multifunctional signal transductors for r apid and e lectronic d etection (RED). This DARPin-RED platform leverages active centrifugal forces and vortex agitation for signal enhancement within a short turnaround time (<30 minutes), achieving highly sensitive (attomolar to femtomolar) detection of Stx2 spiked in food matrices, such as milk, lettuce extract, and ground beef extract, as well as biological fluids, including whole blood, and serum. Additionally, DARPin-RED is capable of detecting multiple Stx2 subtypes without serious background interference, and successful in both differentiating high-toxin-producing E. coli strain (RM5856) from low toxin producer (RM9872) (p < 0.001) and analyzing different bacterial inoculation stages (p = 0.011) from STEC culture within 8 hours post-inoculation. The ability of DARPin-RED to detect Stx2 from food and human specimens at a high sensitivity and specificity using a point-of-care (POC) readout circuit presents a significant advancement for mitigating foodborne outbreaks and effective management of HUS progression.

Numerous potential drug targets, including G-protein-coupled receptors and ion channel proteins, reside on the cell surface as multi-pass membrane proteins. Unfortunately, despite advances in engineering technologies, engineering biologics against multi-pass membrane proteins remains a formidable task. In this review, we focus on the different methods used to prepare/present multi-pass transmembrane proteins for engineering target-specific biologics such as antibodies, nanobodies and synthetic scaffold proteins. The engineered biologics exhibit high specificity and affinity, and have broad applications as therapeutics, probes for cell staining and chaperones for promoting protein crystallization. We primarily cover publications on this topic from the past 10 years, with a focus on the different formats of multi-pass transmembrane proteins. Finally, the remaining challenges facing this field and new technologies developed to overcome a number of obstacles are discussed.