NULISA: a proteomic liquid biopsy platform with attomolar sensitivity and high multiplexing.

The blood proteome holds great promise for precision medicine but poses substantial challenges due to the low abundance of most plasma proteins and the vast dynamic range of the plasma proteome. Here we address these challenges with NUcleic acid Linked Immuno-Sandwich Assay (NULISA™), which improves the sensitivity of traditional proximity ligation assays by ~10,000-fold to attomolar level, by suppressing assay background via a dual capture and release mechanism built into oligonucleotide-conjugated antibodies. Highly multiplexed quantification of both low- and high-abundance proteins spanning a wide dynamic range is achieved by attenuating signals from abundant targets with unconjugated antibodies and next-generation sequencing of barcoded reporter DNA. A 200-plex NULISA containing 124 cytokines and chemokines and other proteins demonstrates superior sensitivity to a proximity extension assay in detecting biologically important low-abundance biomarkers in patients with autoimmune diseases and COVID-19. Fully automated NULISA makes broad and in-depth proteomic analysis easily accessible for research and diagnostic applications.

NULISA: a novel proteomic liquid biopsy platform with attomolar sensitivity and high multiplexing.

The blood proteome holds great promise for precision medicine but poses substantial challenges due to the low abundance of most plasma proteins and the vast dynamic range across the proteome. We report a novel proteomic technology - NUcleic acid Linked Immuno-Sandwich Assay (NULISA™) - that incorporates a dual capture and release mechanism to suppress the assay background and improves the sensitivity of the proximity ligation assay by over 10,000-fold to the attomolar level. It utilizes pairs of antibodies conjugated to DNA oligonucleotides that enable immunocomplex purification and generate reporter DNA containing target- and sample-specific barcodes for a next-generation sequencing-based, highly multiplexed readout. A 200-plex NULISA targeting 124 cytokines and chemokines and 80 other immune response-related proteins demonstrated superior sensitivity for detecting low-abundance proteins and high concordance with other immunoassays. The ultrahigh sensitivity allowed the detection of previously difficult-to-detect, but biologically important, low-abundance biomarkers in patients with autoimmune diseases and COVID-19. Fully automated NULISA addresses longstanding challenges in proteomic analysis of liquid biopsies and makes broad and in-depth proteomic analysis accessible to the general research community and future diagnostic applications.

Structure-function study of poly(sulfobetaine 3,4-ethylenedioxythiophene) (PSBEDOT) and its derivatives.

Poly(3,4-ethylenedioxythiophene) (PEDOT) has been widely studied in recent decades due to its high stability, biocompatibility, low redox potential, moderate band gap, and optical transparency in its conducting state. However, for its long-term in vivo applications, the biocompatibility of PEDOT still needs to be improved. To address this challenge, zwitterionic poly(sulfobetaine 3,4-ethylenedioxythiophene) (PSBEDOT) that contains EDOT backbone with sulfobetaine functional side chains was developed in our previous study. Although PSBEDOT showed great resistance to proteins, cells, and bacteria, it is still not clear how the zwitterionic sulfobetaine side chain affects the electrochemical properties of the polymer and reactivity of the monomer. To achieve better understanding of the structure-function relationships of zwitterionic conducting polymers, we synthesized two derivatives of PSBEDOT, PSBEDOT-4 and PSBEDOT-5, by introducing the alkoxyl spacer between PEDOT backbone and sulfobetaine side chain. The interfacial impedance of PSBEDOT-4 and PSBEDOT-5 was examined by electrochemical impedance spectroscopy and showed significant improvement which is about 20 times lower than PSBEDOT on both gold and indium tin oxide substrates at 1 Hz. In the protein adsorption study, PSBEDOT, PSBEDOT-4 and PSBEDOT-5 exhibited comparable resistance to the fibrinogen solution. All three polymers had low protein adsorption around 3-5% comparing to PEDOT. Additionally, the morphology of PSBEDOT, PSBEDOT-4 and PSBEDOT-5 have been investigated by scanning electron microscopy. We believe that these stable and biocompatible materials can be excellent candidates for developing long-term bioelectronic devices. STATEMENT OF SIGNIFICANCE: To address the challenges associated with existing conducting polymers for bioelectronics, we developed a versatile and high performance zwitterionic conducting material platform with excellent stability, electrochemical, antifouling and controllable antimicrobial/antifouling properties. In this work, we developed two high-performance conducting polymers and systematically investigated how the structure affects their properties. Our study shows we can accurately tune the molecular structure of the monomer to improve the performance of zwitterionic conducting polymer. This zwitterionic conducting polymer platform may dramatically increase the performance and service life of bio-electrochemical devices for many long-term applications, such as implantable biosensing, tissue engineering, wound healing, robotic prostheses, biofuel cell etc., which all require high performance conducting materials with excellent antifouling property/biocompatibility at complex biointerfaces.

Highly sensitive and stable zwitterionic poly(sulfobetaine-3,4-ethylenedioxythiophene) (PSBEDOT) glucose biosensor.

A zwitterionic poly(sulfobetaine-3,4-ethylenedioxythiophene) (PSBEDOT)-based glucose biosensor was fabricated via encapsulating glucose oxidase (GOx) in a one-step electropolymerization method. Integrating conductivity and hydrophilic properties, PSBEDOT provides a great framework for GOx immobilization and stabilization. The anti-fouling, high-sensitivity, and long-term stability properties of PSBEDOT-GOx make it a promising platform for long-term and continuous glucose monitoring.

Ionic Conductivity of Polyelectrolyte Hydrogels.

Polyelectrolytes have many important functions in both living organisms and man-made applications. One key property of polyelectrolytes is the ionic conductivity due to their porous networks that allow the transport of water and small molecular solutes. Among polyelectrolytes, zwitterionic polymers have attracted huge attention for applications that involve ion transport in a polyelectrolyte matrix; however, it is still unclear how the functional groups of zwitterionic polymer side chains affect their ion transport and swelling properties. In this study, zwitterionic poly(carboxybetaine acrylamide), poly(2-methacryloyloxyethyl phosphorylcholine), and poly(sulfobetaine methacrylate) hydrogels were synthesized and their ionic conductivity was studied and compared to cationic, anionic, and nonionic hydrogels. The change of the ionic conductivity of zwitterionic and nonionic hydrogels in different saline solutions was investigated in detail. Zwitterionic hydrogels showed much higher ionic conductivity than that of the widely used nonionic poly(ethylene glycol) methyl ether methacrylate hydrogel in all tested solutions. For both cationic and anionic hydrogels, the presence of mobile counterions led to high ionic conductivity in low salt solutions; however, the ionic conductivity of zwitterionic hydrogels surpassed that of cationic and ionic hydrogels in high salt solutions. Cationic and anionic hydrogels showed much higher water content than that of zwitterionic hydrogels in deionized water; however, the cationic hydrogels shrank significantly with increasing saline concentration. This work provides insight into the effects of polyelectrolyte side chains on ion transport. This can guide us in choosing better polyelectrolytes for a broad spectrum of applications, including bioelectronics, neural implants, battery, and so on.

pH-Sensitive Poly(histidine methacrylamide).

This research reports a synthetic amino acid based zwitterionic poly(histidine methacrylamide) (PHisMA), which possesses switchability among zwitterionic, anionic, and cationic states, pH-dependent antifouling properties, and chelation capability to multivalent metal ions. The PHisMA polymer brush surface shows good antifouling properties to resist protein adsorption and bacterial attachment in its zwitterionic state at pH 5. This study also demonstrates that the solution acidity significantly affects the mechanical properties of PHisMA hydrogels. PHisMA hydrogels show higher viscoelastic properties and lower swelling ratios in the zwitterionic state at pH 4 and pH 5, compared to higher or lower pH conditions. It was discovered that PHisMA can chelate multivalent metal ions, such as Ca(2+), Mg(2+), Cu(2+), Ni(2+) and Fe(3+). This study provides us a better understanding of structure-property relationships of switchable zwitterionic polymers. PHisMA can potentially be adapted for a broad range of applications including wound care, water treatment, bioseparation, coating, drug and gene delivery carriers, etc.

Electroactive poly(sulfobetaine-3,4-ethylenedioxythiophene) (PSBEDOT) with controllable antifouling and antimicrobial properties.

This paper reports a multifunctional zwitterionic poly(sulfobetaine-3,4-ethylenedioxythiophene) (PSBEDOT), which could be facilely synthesized by electropolymerization in aqueous solution. The PSBEDOT surface exhibits excellent conductivity, stability and switchable antifouling/antimicrobial properties. It shows great promise for applications in organic bioelectronics. This new material may significantly increase the performance and service life, minimize the foreign body reaction, improve the biocompatibility and reduce the infection of bio-electronic devices.

Structure-Function Relationships of a Tertiary Amine-Based Polycarboxybetaine.

Zwitterionic polycarboxybetaine (PCB) materials have attracted noticeable interest for biomedical applications, such as wound healing/tissue engineering, medical implants, and biosensors, due to their excellent antifouling properties and design flexibility. Antifouling materials with buffering capability are particularly useful for many biomedical applications. In this work, an integrated zwitterionic polymeric material, poly(2-((2-hydroxyethyl)(2-methacrylamidoethyl)ammonio)acetate) (PCBMAA-1T), was synthesized to carry desired properties (antifouling, switchability and buffering capability). A tertiary amine was used to replace quaternary ammonium as the cation to endow the materials with buffering capability under neutral pH. Through this study, a better understanding on the structure-property relationship of zwitterionic materials was obtained. The tertiary amine cation does not compromise antifouling properties of zwitterionic materials. The amount of adsorbed proteins on PCBMAA-1T polymer brushes is less than 0.8 ng/cm(2) for fibrinogen and 0.3 ng/cm(2) (detection limit of the surface plasmon resonance sensor) for both undiluted blood plasma and serum. It is found that the tertiary amine is favorable to obtain good lactone ring stability in switchable PCB materials. Titration study showed that PCBMAA-1T could resist pH changes under both acidic (pH 1-3) and neutral/basic (pH 7-9) conditions. To the best of our knowledge, such an all-in-one material has not been reported. We believe this material might be potentially used for a variety of applications, including tissue engineering, chronic wound healing and medical device coating.

A versatile microparticle-based immunoaggregation assay for macromolecular biomarker detection and quantification.

The rapid, sensitive and low-cost detection of macromolecular biomarkers is critical in clinical diagnostics, environmental monitoring, research, etc. Conventional assay methods usually require bulky, expensive and designated instruments and relative long assay time. For hospitals and laboratories that lack immediate access to analytical instruments, fast and low-cost assay methods for the detection of macromolecular biomarkers are urgently needed. In this work, we developed a versatile microparticle (MP)-based immunoaggregation method for the detection and quantification of macromolecular biomarkers. Antibodies (Abs) were firstly conjugated to MP through streptavidin-biotin interaction; the addition of macromolecular biomarkers caused the aggregation of Ab-MPs, which were subsequently detected by an optical microscope or optical particle sizer. The invisible nanometer-scale macromolecular biomarkers caused detectable change of micrometer-scale particle size distributions. Goat anti-rabbit immunoglobulin and human ferritin were used as model biomarkers to demonstrate MP-based immunoaggregation assay in PBS and 10% FBS to mimic real biomarker assay in the complex medium. It was found that both the number ratio and the volume ratio of Ab-MP aggregates caused by biomarker to all particles were directly correlated to the biomarker concentration. In addition, we found that the detection range could be tuned by adjusting the Ab-MP concentration. We envision that this novel MP-based immunoaggregation assay can be combined with multiple detection methods to detect and quantify macromolecular biomarkers at the nanogram per milliliter level.

Integrated zwitterionic conjugated poly(carboxybetaine thiophene) as a new biomaterial platform.

An integrated zwitterionic conjugated polymer-based biomaterial platform was designed and studied to address some of the key challenges of conjugated polymers in biomedical applications. This biomaterial platform consists of conjugated polymer backbones and multifunctional zwitterionic side chains. Zwitterionic materials gain electrical conductivity and interesting optical properties through conjugated polymer backbones, and non-biocompatible conjugated polymers obtain excellent antifouling properties, enhanced electrical conductivity, functional groups of bioconjugation and response to environmental stimuli via multifunctional zwitterionic side chains. This platform can potentially be adapted to a wide range of applications (e.g. bioelectronics, tissue engineering and biofuel cell), which require high performance conducting materials with excellent antifouling/biocompatibility at biointerfaces.