Conductive single enzyme nanocomposites prepared by in-situ growth of nanoscale polyaniline for high performance enzymatic bioelectrode.

Enzyme-based electrochemical biosensors hold great promise for applications in health/disease monitoring, drug discovery, and environmental monitoring. However, inherently non-conductive nature of proteinaceous enzymes often hampers effective electron transfer at enzyme-electrode interface, limiting biosensor performance of enzyme bioelectrodes. To address this problem, we present an approach to synthesize polyaniline (PAN)-based conductive single enzyme nanocomposites of glucose oxidase (GOx) (denoted as PAN-GOx). To prevent multimerization of enzymes during nanocomposite synthesis and enable single enzyme wrapping, we activate GOx surface with phenylamine groups based on the programmed diffusion of reactants in the reaction solution. Subsequent in-situ polymerization enables the synthesis of nanoscale conductive PAN layer (∼2.7 nm thickness) grafted from individual GOx molecule. PAN-GOx retains 83% and 74% of its specific activity and catalytic efficiency, respectively, compared to free GOx, while demonstrating a ∼500% improved conductivity. Furthermore, PAN-GOx-based glucose biosensors show an approximately 16- and 3-fold higher sensitivity compared to biosensors prepared by using free GOx and a mixture of PAN and GOx, respectively. This study provides a facile method to fabricate conductive single enzyme nanocomposites with enhanced electron transfer, which can potentially be further modified and/or compounded with conductive materials for demonstrating high performance enzymatic bioelectrodes.

Magnetic hollow fibers of covalent organic frameworks (COF) for pollutant degradation and adsorptive removal.

The shaping of covalent organic frameworks (COFs) from non-processible powder forms into applicable architectures with additional functionality remains a challenge. Using pre-electrospun polymer fibers as a sacrificial template, herein, we report a green synthesis of an architecture in the form of COF hollow fibers with an inner layer of peroxidase-like iron oxide nanoparticles as a catalytic material. When compared to peroxidase-like pristine iron oxide nanoparticles, these COF hollow fibers demonstrate higher catalytic breakdown of crystal violet due to their peroxidase-like activity via advanced oxidation process. Furthermore, as a potential adsorbent, hollow COF fibers exhibit significantly effective adsorption capacity and removal efficiency of organic solvent and oil from water. Because of their magnetic nature, COF hollow fibers can be easily recovered and have exhibited high recycling stability for both catalytic dye degradation and organic solvent removal from water.

Activation of crosslinked lipases in mesoporous silica via lid opening for recyclable biodiesel production.

Lipases catalyze a wide range of industrially important reactions, including the transesterification of triglycerides with alcohols for biodiesel production, and the stabilization of lipases are critical to achieve their recycled uses. Here, nanoscale enzyme reactor (NER) of lipase from Rhizopus oryzae (LP) was prepared via a simple two-step process, comprising of enzyme adsorption into magnetically-separable mesoporous silica and follow-up crosslinking of adsorbed enzymes. In aqueous phase, the specific hydrolysis activity of NER-LP was 4.7 times lower than that of free LP. On the other hand, however, the specific transesterification activity of NER-LP (130.4 µmol/min/mg LP) in organic phase for biodiesel production was 50 times higher than that of free LP (2.6 µmol/min/mg LP). These results reveal that the enzyme crosslinking for the preparation of NER does not interfere with the interfacial activation of LP molecules, opening the lid of LP active site under an optimal hydrophobic environment provided by the combination of organic solvent and mesoporous silica. Magnetic separation and optimized washing protocol facilitated the recycled uses of NER-LP. Highly stable and active NER-LP in magnetically-separable mesoporous silica has demonstrated its great potentials as an environmentally-friendly nanobiocatalyst for various lipase applications, including plasticizers, biosurfactants, functional fatty acids, as well as recyclable biodiesel production.

Development of aptamers for rapid airborne bacteria detection.

Airborne microbes can rapidly spread and cause various infectious diseases worldwide. This necessitates the determination of a fast and highly sensitive detection method. There have been no studies on receptors targeting Citrobacter braakii (C. braakii), a pathogenic bacterium which can exist in the air. In this study, we rapidly isolate an aptamer, a nucleic acid molecule that can specifically bind to C. braakii by centrifugation-based partitioning method (CBPM) reported previously by our groups as omitting the repeated rounds of binding incubation, separation, and amplification that are indispensable for SELEX. The binding affinity and specificity of isolated aptamers are checked using bacteria in liquid culture and recollection solution from aerosolized bacteria. Recollection solutions of the recovered bacteria are obtained by nebulizing, drying, and recapturing with a biosampler. The CB-5 aptamer shows high affinity and specificity for C. braakii (Kd: 16.42 in liquid culture and 26.91 nM in recollection from aerosolized sample). Our results indicate the current protocol can be employed for the rapid development of reliable diagnostic receptors targeting airborne bacteria.

Controlled growth of redox polymer network on single enzyme molecule for stable and sensitive enzyme electrode.

The electrochemical applications of enzymes are often hampered by poor enzyme stability and low electron conductivity. In this work, a novel enzyme nanogel based on atom transfer radical polymerization (ATRP) has been developed for highly sensitive detection of glucose based on ferrocene (Fc) embedded in crosslinked polymer network nanogel. Enzyme surfaces are successively modified with Br initiator, and then in situ atom transfer radical polymerization (ATRP) was performed to build up crosslinked polyacrylamide network. The resulting single enzyme nanogel (ATRP-SEG) is uniform in size fairly. ATRP-SEG reveals bi-phasic inactivation, and the half-life of stable ATRP-SEG after 18-day incubation at 50 °C is 47 days, which is 197 times longer than that of free Gox (5.7 h). By introducing a ferrocene (Fc) containing redox polymer, poly(acrylamide-co-vinylferrocene), the half-life of Fc-ATRP-SEG after 18-day incubation at 50 °C is 49 days. Fc-ATRP-SEG is used for preparation of glucose-sensing electrode, and the sensitivity of Fc-ATRP-SEG electrode is 111 µA cm-2 mM-1, which is 366 and 1270 times higher than those of free GOx (0.303 µA cm-2 mM-1) and ATRP-SEG (0.0874 µA cm-2 mM-1), respectively. Fc-ATRP-SEG electrode maintained 90% of initial current density under 4 °C storage condition and repetitive usages every day for 7 days. Even the electrode repeatedly used in continuous harsh condition (250 rpm, room temperature), the current density maintained 96% after 12 h incubation at operational condition.

Precipitation-based microscale enzyme reactors coupled with porous and adhesive elastomer for effective bacterial decontamination and membrane antifouling on-demand.

Bacterial contamination of water environments can cause various troubles in various areas. As one of potential solutions, we develop enzyme-immobilized elastomer, and demonstrate the uses of enzyme reactions on-demand for effective microbial decontamination and antifouling. Asymmetrically-structured elastomer is prepared by combining two polydimethylsiloxane (PDMS) layers with different degrees of crosslinking: highly-crosslinked and lightly-crosslinked PDMS layers. At the surface of highly-crosslinked PDMS layer, porous structure with average diameter of 842 nm is formed by dissolving pre-packed and entrapped latex beads. Lightly-crosslinked PDMS on the other side, due to its adhesive nature, enables iterative attachments on various materials under either dry or wet condition. Glucose oxidase (GOx) is immobilized by using the pores at the surface of highly-crosslinked PDMS matrix via a ship-in-a-bottle protocol of precipitation-based microscale enzyme reactor (p-MER), which consists of GOx adsorption, precipitation and chemical crosslinking (EAPC). As a result, crosslinked enzyme aggregates (CLEAs) of GOx not only are well entrapped within many pores of highly-crosslinked PDMS layer (ship-in-bottle) but also cover the external surface of matrix, both of which are well connected together. Highly-interconnected network of CLEAs themselves effectively prevents enzyme leaching, which shows the 25% residual activity of GOx under shaking at 200 rpm for 156 days after 48% initial drop of loosely-bound p-MER after 4 days. In presence of glucose, the underwater attachment of biocatalytic elastomer demonstrates the generation of hydrogen peroxide via p-MER-catalyzed glucose oxidation, exhibiting effective biocidal activities against both gram-positive S. aureus and gram-negative E. coli. Adhesion-induced GOx-catalyzed reaction also alleviates the biofouling of membrane, suggesting its extendibility to various engineering systems being suffered by biofouling. This study of biocatalytic elastomer has demonstrated its new opportunities for the facile and on-demand enzyme-catalyzed reactions in various environmental applications, such as bactericidal treatment, water treatment/purification, and pollutant degradation.

Recent advances in multifunctional nanomaterials for photothermal-enhanced Fenton-based chemodynamic tumor therapy.

Photothermal (PT)-enhanced Fenton-based chemodynamic therapy (CDT) has attracted a significant amount of research attention over the last five years as a highly effective, safe, and tumor-specific nanomedicine-based therapy. CDT is a new emerging nanocatalyst-based therapeutic strategy for the in situ treatment of tumors via the Fenton reaction or Fenton-like reaction, which has got fast progress in recent years because of its high specificity and activation by endogenous substances. A variety of multifunctional nanomaterials such as metal-, metal oxide-, and metal-sulfide-based nanocatalysts have been designed and constructed to trigger the in situ Fenton or Fenton-like reaction within the tumor microenvironment (TME) to generate highly cytotoxic hydroxyl radicals (•OH), which is highly efficient for the killing of tumor cells. However, research is still required to enhance the curative outcomes and minimize its side effects. Specifically, the therapeutic efficiency of certain CDTs is still hindered by the TME, including low levels of endogenous hydrogen peroxide (H2O2), overexpression of reduced glutathione (GSH), and low catalytic efficacy of Fenton or Fenton-like reactions (pH 5.6-6.8), which makes it difficult to completely cure cancer using monotherapy. For this reason, photothermal therapy (PTT) has been utilized in combination with CDT to enhance therapeutic efficacy. More interestingly, tumor heating during PTT not only causes damage to the tumor cells but can also accelerate the generation of •OH via the Fenton and Fenton-like reactions, thus enhancing the CDT efficacy, providing more effective cancer treatment when compared with monotherapy. Currently, synergistic PT-enhanced CDT using multifunctional nanomaterials with both PT and chemodynamic properties has made enormous progress in cancer theranostics. However, there has been no comprehensive review on this subject published to date. In this review, we first summarize the recent progress in PT-enhanced Fenton-based CDT for cancer treatment. We then discuss the potential and challenges in the future development of PT-enhanced Fenton-based nanocatalytic tumor therapy for clinical application.

Carbonic anhydrase for CO2 capture, conversion and utilization.

Carbonic anhydrase (CA) enzymes, catalyzing the CO2 hydration at a high turnover number, can be employed in expediting CO2 capture, conversion and utilization to aid in carbon neutrality. Despite extensive research over the last decade, there remain challenges in CA-related technologies due to poor stability and suboptimal use of CAs. Herein, we discuss recent advances in CA stabilization by protein engineering and enzyme immobilization, and shed light on state-of-the-art of in vitro and in vivo CA-mediated CO2 conversion for improved production of value-added chemicals using CO2 as a feedstock.

High Oxygen and Water-Vapor Transmission Rate and In Vitro Cytotoxicity Assessment with Illite-Polyethylene Packaging Films.

This work reports the preparation of a ceramic hybrid composite film with illite and polyethylene (illite-PE), and the evaluation of the freshness-maintaining properties such as oxygen transmission rate (OTR), water vapor transmission rate (WVTR), tensile strength, and in vitro cytotoxicity. The particle size of the illite material was controlled to within 10 µm. The illite-PE masterbatch and film were prepared using a twin-screw extruder and a blown film maker, respectively. The dispersity and contents of illite material in each masterbatch and composite film were analyzed using a scanning electron microscope (SEM) and thermogravimetric analyzer (TGA). In addition, the OTR and WVTR of the illite-PE composite film were 8315 mL/m2·day, and 13.271 g/m2·day, respectively. The in vitro cytotoxicity of the illite-PE composite film was evaluated using L929 cells, and showed a cell viability of more than 92%. Furthermore, the freshness-maintaining property was tested for a packaging application with bananas; it was found to be about 90%, as indicated by changes in the color of the banana peel, after 12 days.

Stabilized and Immobilized Carbonic Anhydrase on Electrospun Nanofibers for Enzymatic CO2 Conversion and Utilization in Expedited Microalgal Growth.

Carbonic anhydrases convert CO2 to bicarbonate at a high turnover rate up to 106 s-1, but their actual applications in CO2 conversion processes are hampered by their poor stability. This study reports highly loaded and stabilized bovine carbonic anhydrase (bCA) upon being immobilized onto electrospun polymer nanofibers in the form of enzyme precipitate coating (EPC). The EPC protocol, consisting of enzyme covalent attachment, precipitation, and cross-linking, maintained 65.3% of initial activity even after being incubated in aqueous solution at room temperature under shaking at 200 rpm for 868 days. EPC also showed strong resistance to the treatment of the metal chelation agent, ethylenediaminetetraacetic acid, and molecular dynamic simulation was carried out to elucidate the prevention of metal leaching from the active site of bCA upon being cross-linked in the form of EPC. Highly stable EPC with high bCA loading was employed for the conversion of bubbling CO2 to bicarbonate, and the bicarbonate solution was utilized as a carbon source for expedited microalgae growth in a separate bioreactor. The addition of EPC in the bubbling CO2 reactor resulted in 134 and 231% accelerated microalgae growths compared to the controls with and without 25 mM sodium bicarbonate, respectively. EPC with high enzyme loading and unprecedentedly successful stabilization of enzyme stability has a great potential to be used for the development of various enzyme-mediated CO2 conversion and utilization technologies.

Crowding and confinement effects on enzyme stability in mesoporous silicas.

To understand the protein functions within a cell, where proteins exist in an extremely crowded and confined state, various modeling and experimental methods have been proposed. Here, we propose a new experimental approach to modulate the macromolecular crowding and/or confinement effects by using mesoporous silicas with two different pore structures. SBA-15 and MSU-F with linear and mesocellular pore structures, respectively, were used to adsorb a model enzyme, glucose oxidase (GOx), in various concentrations ranging from 3 to 430 mg ml-1. The concentration of adsorbed GOx in the mesopores, representing the degree of crowding, showed a good correlation with thermal enzyme stability. Interestingly, the increase of thermal stability as a function of macromolecular crowding showed different correlations depending on the pore structure of mesoporous silicas. It represents that combination of crowding and confinement effects can promote different microenvironments for enzyme molecules, while mesoporous silicas can impose controlled crowding and confinement effects on enzymes due to their uniform and tunable pore structures. It is anticipated that this new and simple approach can provide a tool to elucidate crowding and confinement effects on the protein functions, including its stability in vivo, because the mesopore environments could mimic the real macromolecular cell system under crowding.

Glucose oxidase-copper hybrid nanoflowers embedded with magnetic nanoparticles as an effective antibacterial agent.

Bacterial contamination causes various problems ranging from bacterial infection to biofouling. As an effective and non-toxic agent for bacterial de-contamination, glucose oxidase (GOx)-copper hybrid nanoflowers embedded with amine-functionalized magnetic nanoparticles (NH2-MNPs), called 'MNP-GOx NFs', are developed. Positively-charged NH2-MNPs and negatively-charged GOx molecules are first interacted via electrostatic attraction which can be controlled by changing the buffer pH, and the follow-up addition of copper(II) sulfate leads to blooming of nanoflowers (MNP-GOx NFs) after incubation at room temperature for 3 days. MNP-GOx NFs show effective antibacterial activity by generating H2O2 from GOx-catalyzed glucose oxidation. For example, 99.9% killings of Staphylococcus aureus and Escherichia coli are achieved after 3 h treatment of 106/mL cells with 0.2 and 3.0 mg/mL MNP-GOx NFs, respectively, revealing that Gram-positive S. aureus with mono-layer membrane system is more vulnerable to the treatment of MNP-GOx NFs than Gram-negative E. coli with two-layer membrane system. MNP-GOx NFs can maintain 97% of bactericidal activity even after recycled uses by magnetic separation for eight times iterative bacterial killings. Finally, MNP-GOx NFs are employed for the fabrication of antibacterial gauzes. MNP-GOx NFs have also opened up a great potential for their applications in biosensors, biofuel cells and bioconversion as well as bacterial de-contamination.

Modular Assembly of Unique Chimeric Lytic Enzymes on a Protein Scaffold Possessing Anti-Staphylococcal Activity.

Lytic enzymes have been considered as potential alternatives to antibiotics. These enzymes, particularly those that target Gram-positive bacteria, consist of modular cell wall-binding and catalytic domains, which can be shuffled with those of other lytic enzymes to produce unnatural chimeric enzymes. In this work, we report the in vitro shuffling of two different modular domains using a protein self-assembly methodology. Catalytic domains (CD) and cell wall-binding domains (BD) from the bacteriocin lysostaphin (Lst) and a putative autolysin from Staphylococcus aureus (SA1), respectively, were genetically site-specifically biotinylated and assembled with streptavidin to generate 23 permuted chimeras. The specific assembly of a CD (3 equiv) and a BD (1 equiv) from Lst and SA1, respectively [CDL-BDS (3:1)], on a streptavidin scaffold yielded high lytic activity against S. aureus (at least 5.6 log reduction), which was higher than that obtained with either native Lst or SA1 alone. Moreover, at 37 °C, the initial rate of cell lysis was over 3-fold higher than that with free Lst, thereby revealing the unique catalytic properties of the chimeric proteins. In vitro self-assembly of functional domains from modular lytic enzymes on a protein scaffold likely expands the repertoire of bactericidal enzymes with improved activities.

Enzyme-Immobilized Chitosan Nanoparticles as Environmentally Friendly and Highly Effective Antimicrobial Agents.

Highly effective and minimally toxic antimicrobial agents have been prepared by immobilizing glucose oxidase (GOx) onto biocompatible chitosan nanoparticles (CS-NPs). CS-NPs were prepared via ionotropic gelation and used for the immobilization of GOx via approaches of covalent attachment (CA), enzyme coating (EC), enzyme precipitate coating (EPC), and magnetic nanoparticle-incorporated EPC (Mag-EPC). EPC represents an approach consisting of enzyme covalent attachment, precipitation, and cross-linking, with CA and EC being control samples while Mag-EPC was prepared by mixing magnetic nanoparticles (Mag) with enzymes during the preparation of EPC. The GOx activities of CA, EC, EPC, and Mag-EPC were 8.57, 17.7, 219, and 247 units/mg CS-NPs, respectively, representing 26 and 12 times higher activity of EPC than those of CA and EC, respectively. EPC improved the activity and stability of GOx and led to good dispersion of CS-NPs, while Mag-EPC enabled facile magnetic separation. To demonstrate the expandability of the EPC approach to other enzymes, bovine carbonic anhydrase was also employed to prepare EPC and Mag-EPC samples for their characterizations. In the presence of glucose, EPC of GOx generated H2O2 in situ, which effectively inhibited the proliferation of Staphylococcus aureus in both suspended cultures and biofilms, thereby demonstrating the potential of EPC-GOx as environmentally friendly and highly effective antimicrobial materials.

Tyrosinase-immobilized CNT based biosensor for highly-sensitive detection of phenolic compounds.

Highly sensitive phenol biosensor was developed by using well-dispersed carbon nanotubes (CNTs) in enzyme solution and adding CNTs in enzyme electrodes. First, the intact CNTs were dispersed in aqueous tyrosinase (TYR) solution, and TYR molecules were precipitated and crosslinked to prepare the sample of enzyme adsorption, precipitation and crosslinking (EAPC). EAPC exhibited 10.5- and 5.4-fold higher TYR activity per mg of CNTs as compared to enzyme adsorption (EA) and enzyme adsorption/crosslinking (EAC), respectively. EAPC retained 29% of its initial activity after incubation at 40 °C for 128 h, while EA and EAC showed no residual activities, respectively. In biosensing a model phenolic compound of catechol, the sensitivities of EA, EAC and EAPC electrodes on glassy carbon electrode (GCE) were 34, 281 and 675 µA/mM/cm2, respectively. When 90 w/w% CNTs were added to the enzyme electrodes, the sensitivities of EA, EAC, and EAPC electrodes were 146, 427, and 1160 µA/mM/cm2, respectively, and the EAPC electrode showed a 2.3-fold increase in sensitivity upon CNT addition. Catechol and phenol could also be detected by EAPC on the screen-printed electrode (SPE), with sensitivities of 1340 and 1170 µA/mM/cm2, respectively. The sensitivity of EAPC-SPE for phenol detection in the effluent from real municipal wastewater treatment plant was 1100 µA/mM/cm2. The sensitivity of EAPC-SPE retained 74% of its initial sensitivity after incubation at 40 °C for 12 h. The combination of EAPC immobilization and CNT addition has great potential for application in the development of sensitive enzyme biosensors for various analytes and phenols in water environments.

Magnetic Nanoparticles-Embedded Enzyme-Inorganic Hybrid Nanoflowers with Enhanced Peroxidase-Like Activity and Substrate Channeling for Glucose Biosensing.

It is reported that glucose oxidase (GOx)-copper hybrid nanoflowers embedded with Fe3 O4 magnetic nanoparticles (MNPs) exhibit superior peroxidase-mimicking activity as well as substrate channeling for glucose detection. This is due to the synergistic integration of GOx, crystalline copper phosphates and MNPs being in close proximity within the nanoflowers. The preparation of MNP-embedded GOx-copper hybrid nanoflowers (MNPs-GOx NFs) begins with the facile conjugation of amine-functionalized MNPs with GOx molecules via electrostatic attraction, followed by the addition of copper sulfate that leads to full blooming of the hybrid nanoflowers. In the presence of glucose, the catalytic action of GOx entrapped in the nanoflowers generates H2 O2 , which is subsequently used by peroxidase-mimicking MNPs and copper phosphate crystals, located close to GOx molecules, to convert Amplex UltraRed substrate into a highly fluorescent product. Using this strategy, the target glucose is successfully determined with excellent selectivity, stability, and magnetic reusability. This biosensor based on hybrid nanoflowers also exhibits a high degree of precision and reproducibility when applied to real human blood samples. Such novel MNP-embedded enzyme-inorganic hybrid nanoflowers have a great potential to be expanded to any oxidases, which will be highly beneficial for the detection of various other clinically important target molecules.

Effect of Surface and Bulk Properties of Mesoporous Carbons on the Electrochemical Behavior of GOx-Nanocomposites.

Biofuel cell (BFC) electrodes are typically manufactured by combining enzymes that act as catalysts with conductive carbon nanomaterials in a form of enzyme-nanocomposite. However, a little attention has been paid to effects of the carbon nanomaterials' structural properties on the electrochemical performances of the enzyme-nanocomposites. This work aims at studying the effects of surface and bulk properties of carbon nanomaterials with different degrees of graphitization on the electrochemical performances of glucose oxidase (GOx)-nanocomposites produced by immobilizing GOx within a network of carbon nanopaticles. Two types of carbon nanomaterials were used: graphitized mesoporous carbon (GMC) and purified mesoporous carbon (PMC). Graphitization index, surface functional groups, hydrophobic properties, and rate of aggregation were measured for as-received and acid-treated GMC and PMC samples by using Raman spectrometry, X-ray photoelectron spectroscopy (XPS), contact angle measurement, and dynamic light scattering (DLS), respectively. In addition to these physical property characterizations, the enzyme loading and electrochemical performances of the GOx-nanocomposites were studied via elemental analysis and cyclic voltammetry tests, respectively. We also fabricated BFCs using our GOx-nanocomposite materials as the enzyme anodes, and tested their performances by obtaining current-voltage (IV) plots. Our findings suggest that the electrochemical performance of GOx-nanocomposite material is determined by the combined effects of graphitization index, electrical conductivity and surface chemistry of carbon nanomaterials.

Lag Time Spectrophotometric Assay for Studying Transport Limitation in Immobilized Enzymes.

Enzymes are promising catalysts for bioprocessing. For instance, the enzymatic capture of CO2 using carbonic anhydrase (CA) is a carbon capture approach that allows obtaining bicarbonate (HCO3 -) with no high-energy input required. However, application in a commercially viable biotechnology requires sufficient enzymatic lifetime. Although enzyme stabilization can be achieved by different immobilization techniques, most of them are not commercially viable because of transport limitations induced by the immobilization method. Therefore, it is necessary to develop assays for evaluating the role of immobilization on transport limitations. Herein, we describe the development of a fast and reproducible assay for screening immobilized CA by means of absorbance measurement using a computer-controlled microplate reader in stop-flow format. The automated assay allowed minimizing the required volume for analysis to 120 µL. We validated the assay by determining lag times and activities for three immobilization techniques (modified Nafion, hydrogels, and enzyme precipitates), of which linear polyethyleneimine hydrogel showed outstanding performance for CA immobilization.

Sensitive multiplex detection of whole bacteria using self-assembled cell binding domain complexes.

Detecting bacterial cells at low levels is critical in public health, the food industry and first response. Current processes typically involve laborious cell lysis and genomic DNA extraction to achieve 100-1000 CFU mL-1 levels for detecting gram-positive bacteria. As an alternative to DNA-based methods, cell wall binding domains (CBDs) derived from lysins having a modular structure with an N-terminal catalytic domain and a C-terminal CBD, can be used to detect bacterial pathogens as a result of their exceptionally specific binding to target bacteria with great avidity. We have developed a highly sensitive method for multiplex detection of whole bacterial cells using self-assembled CBD complexes. Self-assembled CBD-SA-reporter complexes were generated using streptavidin (SA), biotin-CBDs, and biotinylated reporters, such as glucose oxidase (GOx) and specific DNA sequences. The simultaneous detection of three test bacteria, Staphylococcus aureus, Bacillus anthracis-Sterne, and Listeria innocua cells in PBS could be accomplished with a 96-well plate-based sandwich method using CBD-SA-GOx complex-coupled spectrophotometric assay to achieve a detection limit of >100 CFU mL-1. To achieve greater detection sensitivity, we used CBD-SA-DNA complexes and qPCR of specific DNA barcodes selectively bound to the surface of target bacterial cells, which resulted in a detection sensitivity as low as 1-10 CFU mL-1 without cross-reactivity. This sensitive multiplex detection of bacterial pathogens using both CBD-SA-GOx and CBD-SA-DNA complexes has the potential to be quickly combined with point-of-care compatible diagnostics for the rapid detection of pathogens in test samples.