Chao Wang

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.

DNA origami information storage is a promising alternative to silicon-based data storage, offering a molecular cryptography technique concealing information within DNA origami. Routing, sliding, and interlacing staple strands lead to a large 700-bit key size. Practical DNA data storage requires high information density, robust security, and accurate and rapid information retrieval. Consequently, advanced readout techniques and large encryption key sizes are essential. Here, we report an enhanced DNA origami cryptography protocol in 2D and 3D DNA origami, increasing the encryption key size. We employ all-DNA-based steganography with fast readout through high-speed DNA-PAINT super-resolution imaging. By combining DNA-PAINT data with unsupervised clustering, we achieve an accuracy of up to 89%, despite the flexibility in the 3D DNA origami shown by oxDNA simulation. Furthermore, we propose criteria that ensure complete information retrieval for the DNA origami cryptography. Our findings show that DNA-based cryptography is a secure and versatile solution for storing information.

The increasing demand for high‐capacity data storage continues to drive innovation in memory technologies. DNA has recently emerged as a promising medium due to its exceptionally high data density and long‐term stability. Here, a DNA‐based data storage and readout scheme is presented that leverages sapphire‐supported, low‐noise solid‐state nanopores for reliable detection of nanostructured DNA. Data are encoded onto four‐helix bundle (4HB) DNA structures, enabling flexible programmability and secure readout. The sapphire‐supported nanopores demonstrate a high signal‐to‐noise ratio (≈22) under a 250 kHz filtering frequency, allowing precise signal discrimination during readout. Using a decision‐tree supervised learning algorithm, up to ≈93% of encoded messages are accurately classified, demonstrating the feasibility of letter and message encoding for information retrieval. This work highlights the potential of integrating DNA nanostructures with low‐noise nanopore technology for high‐density, secure, and scalable data storage applications.

Major challenges remain to precisely detect low-abundance proteins rapidly and cost-effectively from diverse biofluids. Here, we present a gold nanoparticle (AuNP)-supported, rapid electronic detection (NasRED) platform with subfemtomolar sensitivity and high specificity. Surface-functionalized AuNPs act as multivalent detectors to recognize target antigens and antibodies through high-affinity binding, subsequently forming aggregates precipitated in a microcentrifuge tube and producing a solution color change. The residual floating AuNPs' optical extinction is digitized using customized circuitry incorporating inexpensive optoelectronics and feedback mechanisms for stabilized readout. NasRED introduces active fluidic forces through engineered centrifugation and vortex agitation, effectively promoting low-concentration protein detection and accelerating signal transduction. Using SARS-CoV-2 as a demonstration, NasRED enables detection of both antibodies and antigens from a small sample volume (6 μL), distinguishes the viral antigens from those of human coronaviruses, and delivers test results in 3000 times more sensitive than enzyme-linked immunosorbent assay (ELISA), ∼76 aM (11 fg/mL) in human pooled serum and in the femtomolar range in diluted whole blood. For nucleocapsid protein detection, NasRED LoDs are ∼190 aM (10 fg/mL) in human saliva and ∼2 fM (100 fg/mL) in nasal fluid. Unlike centralized platforms, NasRED is a one-pot, in-solution assay without the needs for washing, labeling, expensive instrumentation, or highly trained operators. With low reagent costs and a compact system footprint, this modular digital platform is well-suited for accurate, near-patient diagnosis and screening of a wide range of infectious and chronic diseases.

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.

Thrombospondin-2 (THBS2) is a prevailing prognostic biomarker implicated in different cancer types, such as deadly colorectal, pancreas, and triple-negative breast cancers. While the current methods for cancer-relevant protein detection, such as enzyme-linked immunosorbent assay (ELISA), mass spectrometry, and immunohistochemistry, are feasible at advanced stages, they have shortcomings in sensitivity, specificity, and accessibility, particularly at low concentrations in complex biological fluids for early detection. Here, we propose and demonstrate a modular, in-solution assay design concept, Nanoparticle-Supported Rapid Electronic Detection (NasRED), as a versatile cancer screening and diagnostic platform. NasRED utilizes antibody-functionalized gold nanoparticles (AuNPs) to capture target proteins from a minute amount of sample (<10 µL) and achieve optimal performance with a short assay time by introducing active fluidic forces that act to promote biochemical reaction and accelerate signal transduction. This rapid (15 min) process serves to form AuNP clusters upon THBS2 binding and subsequently precipitate such clusters, resulting in color modulation of the test tubes that is dependent on the THBS2 concentration. Finally, a semiconductor-based, portable electronic device is used to digitize the optical signals for the sensitive detection of THBS2. High sensitivity (femtomolar level) and a large dynamic range (five orders of magnitude) are obtained to analyze THBS2 spiked in PBS, serum, whole blood, saliva, cerebrospinal fluids, and synovial fluids. High specificity is also preserved in differentiating THBS2 from other markers such as cancer antigen (CA) 19-9 and bovine serum albumin (BSA). This study highlights NasRED’s potential to enhance cancer prognosis and screening by offering a cost-effective, accessible, and minimally invasive solution.

The measurement of neutralizing immune responses to viral infection is essential, given the heterogeneity of human immunity and the emergence of new virus strains. However, neutralizing antibody (nAb) assays often require high-level biosafety containment, sophisticated instrumentation, and long detection times. Here, as a proof-of-principle, we designed a nanoparticle-supported, rapid, electronic detection (NasRED) assay to assess the neutralizing potency of monoclonal antibodies (mAbs) against SARS-CoV-2. The gold nanoparticles (AuNPs) coated with human angiotensin-converting enzyme 2 (ACE2) protein as nAb potency reporters were mixed with the mAbs to be tested, as well as streptavidin-conjugated multivalent spike (S) protein or their receptor binding domains (RBD). High-affinity and ACE2-competitive nAbs alter the S (or RBD)-to-ACE2 binding level and modulate AuNP cluster formation and precipitation. The amount of free-floating AuNP reporters is quantified by a semiconductor-based readout system that measures the AuNPs’ optical extinction, producing nAb signals that can differentiate SARS-CoV-2 variants (Wuhan-Hu-1, Gamma, and Omicron). The modular design nature, short assay time (less than 30 minutes), and portable and inexpensive readout system make this NasRED-nAb assay applicable to measuring vaccine potency, immune responses to infection, and the efficacy of antibody-based therapies.

Major challenges remain to precisely detect low-abundance proteins from diverse biofluids in a rapid and cost-effective manner. Here we present a gold nanoparticle (AuNP)-supported, rapid electronic detection (NasRED) platform with sub-femtomolar sensitivity and high specificity. Surface-functionalized AuNPs act as multivalent detectors to recognize target antigens and antibodies through high-affinity binding, subsequently forming aggregates precipitated in a microcentrifuge tube and producing a solution color change. The optical extinction of residual floating AuNPs is digitized using a customized circuitry incorporating inexpensive optoelectronic elements and feedback mechanisms for stabilized readout. Uniquely, NasRED introduces active fluidic forces through engineered centrifugation and vortex agitation, effectively promoting protein detection at low concentrations and accelerating signal generation. Using SARS-CoV-2 as a demonstration, NasRED enables detection of both antibodies and antigens from a small sample volume (6 µL), distinguishes the viral antigens from those of human coronaviruses, and delivers test results in a short time (as fast as 3,000 times more sensitive than Enzyme-Linked Immunosorbent Assay (ELISA), ∼76 aM (11 fg/mL) in human pooled serum and in the femtomolar range in diluted whole blood. For nucleocapsid protein detection, NasRED LoDs are ∼190 aM (10 fg/mL) in human saliva and ∼2 fM (100 fg/mL) in nasal fluid. Unlike laboratory-based ELISA platforms, NasRED is a one-pot, in-solution assay that eliminates the needs for washing, labeling, expensive instrumentation or highly trained operators. With low reagent costs and a compact system footprint, this modular digital platform is well-suited for accurate, near-patient diagnosis and screening of a wide range of infectious and chronic diseases.