Juhong Chen

Detection of Escherichia coli in Drinking Water Using T7 Bacteriophage-Conjugated Magnetic Probe

In this study, we demonstrate a bacteriophage (phage)-based magnetic separation scheme for the rapid detection of Escherichia coli (E. coli) in drinking water. T7 phage is a lytic phage with a broad host range specificity for E. coli. Our scheme was as follows: (1) T7 bacteriophage-conjugated magnetic beads were used to capture and separate E. coli BL21 from drinking water; (2) subsequent phage-mediated lysis was used to release endemic β-galactosidase (β-gal) from the bound bacterial cells; (3) the release of β-gal was detected using chlorophenol red-β-d-galactopyranoside (CRPG), a colorimetric substrate which changes from yellow to red in the presence of β-gal. Using this strategy, we were able to detect E. coli at a concentration of 1 × 10(4) CFU·mL(-1) within 2.5 h. The specificity of the proposed magnetic probes toward E. coli was demonstrated against a background of competing bacteria. By incorporating a pre-enrichment step in Luria-Bertani (LB) broth supplemented with isopropyl β-d-thiogalactopyranoside (IPTG), we were able to detect 10 CFU·mL(-1) in drinking water after 6 h of pre-enrichment. The colorimetric change can be determined either by visual observation or with a reader, allowing for a simple, rapid quantification of E. coli in resource-limited settings.

Nanoimprinted Patterned Pillar Substrates for Surface-Enhanced Raman Scattering Applications

A pragmatic method to deposit silver nanoparticles on polydopamine-coated nanoimprinted pillars for use as surface-enhanced Raman scattering (SERS) substrates was developed. Pillar arrays consisting of poly(methyl methacrylate) (PMMA) that ranged in diameter from 300 to 500 nm were fabricated using nanoimprint lithography. The arrays had periodicities from 0.6 to 4.0 μm. A polydopamine layer was coated on the pillars in order to facilitate the reduction of silver ions to create silver nucleation sites during the electroless deposition of sliver nanoparticles. The size and density of silver nanoparticles were controlled by adjusting the growth time for the optimization of the SERS performance. The size of the surface-adhered nanoparticles ranged between 75 and 175 nm, and the average particle density was ∼30 particles per μm(2). These functionalized arrays had a high sensitivity and excellent signal reproducibility for the SERS-based detection of 4-methoxybenzoic acid. The substrates were also able to allow the SERS-based differentiation of three types of bacteriophages (λ, T3, and T7).

Colorimetric Detection of Escherichia coli Based on the Enzyme‐Induced Metallization of Gold Nanorods

A novel enzyme-induced metallization colorimetric assay is developed to monitor and measure beta-galactosidase (β-gal) activity, and is further employed for colorimetric bacteriophage (phage)-enabled detection of Escherichia coli (E. coli). This assay relies on enzymatic reaction-induced silver deposition on the surface of gold nanorods (AuNRs). In the presence of β-gal, the substrate p-aminophenyl β-d-galactopyranoside is hydrolyzed to produce p-aminophenol (PAP). Reduction of silver ions by PAP generates a silver shell on the surface of AuNRs, resulting in the blue shift of the longitudinal localized surface plasmon resonance peak and multicolor changes of the detection solution from light green to orange-red. Under optimized conditions, the detection limit for β-gal is 128 pM, which is lower than the conventional colorimetric assay. Additionally, the assay has a broader dynamic range for β-gal detection. The specificity of this assay for the detection of β-gal is demonstrated against several protein competitors. Additionally, this technique is successfully applied to detect E. coli bacteria cells in combination with bacteriophage infection. Due to the simplicity and short incubation time of this enzyme-induced metallization colorimetric method, the assay is well suited for the detection of bacteria in low-resource settings.

Highly sensitive and selective detection of nitrite ions using Fe3O4@SiO2/Au magnetic nanoparticles by surface-enhanced Raman spectroscopy.

A novel and pragmatic method was developed to detect the concentration of nitrite ions using Fe3O4@SiO2/Au magnetic nanoparticles (MNPs) by surface-enhanced Raman scattering (SERS). The as-prepared bifunctional nanocomposites can be used to simultaneously purify target molecules using external magnetic field and produce Raman fingerprint spectrum with trace level of target molecules. In acidic media, 4-aminothiophenol (4-ATP) molecules conjugated on Fe3O4@SiO2/Au MNPs were triggered by nitrite ions to form azo bonds, resulting in three new marker peaks on the SERS spectrum. Under optimized conditions, the detection limit based on the three marker peaks were 15.63, 13.69, and 17.77μM, which was much lower than the maximum NO2(-) concentration of 1.0mgL(-1) (71.4μM) allowed in drinking water as defined by U.S. Environmental Protection Agency (EPA). The specificity of this proposed method to detect nitrite ions was demonstrated using common ions as competitors. In addition, the SERS-based technique was successfully employed to detect nitrite ions in pond water, a synthetic urine solution, and pickle brine. Considering its good sensitivity and selectivity, the detection method is well suited for the detection of nitrite ions in real samples without pretreatment steps.

Electrochemical Detection of Escherichia coli from Aqueous Samples Using Engineered Phages

In this study, an enzyme-based electrochemical method was developed for the detection of Escherichia coli (E. coli) using the T7 bacteriophages engineered with lacZ operon encoding for beta-galactosidase (β-gal). The T7lacZ phages can infect E. coli, and have the ability to trigger the overexpression of β-gal during the infection of E. coli. The use of the engineered phages resulted in a more sensitive detection of E. coli by (1) overexpression of β-gal in E. coli during the specific infection and (2) release of the endogenous intracellular β-gal from E. coli following infection. The endogenous and phage-induced β-gal was detected using the electrochemical method with 4-aminophenyl-β-galactopyranoside (PAPG) as a substrate. The β-gal catalyzed PAPG to an electroactive species p-aminophenol (PAP) which could be monitored on an electrode. The electrochemical signal was proportional to the concentration of E. coli in the original sample. We demonstrated the application of our strategy in aqueous samples (drinking water, apple juice, and skim milk). Using this method, we were able to detect E. coli at the concentration of approximately 105 CFU/mL in these aqueous samples in 3 h and 102 CFU/mL after 7 h. This strategy has the potential to be extended to detect different bacteria using specific bacteriophages engineered with gene encoding for appropriate enzymes.

Hybrid Antifouling and Antimicrobial Coatings Prepared by Electroless Co-Deposition of Fluoropolymer and Cationic Silica Nanoparticles on Stainless Steel: Efficacy against Listeria monocytogenes.

Controlling formation, establishment, and proliferation of microbial biofilms on surfaces is critical for ensuring public safety. Herein, we report on the synthesis of antimicrobial nanoparticles and their co-deposition along with fluorinated nanoparticles during electroless nickel plating of stainless steel. Plating bath composition is optimized to ensure sufficiently low surface energy to resist fouling and microbial adhesion as well as to exert significant (>99.99% reduction) antimicrobial activity against Listeria monocytogenes. The resulting coatings present hybrid antifouling and antimicrobial character, can be applied onto stainless steel, and do not rely on leaching or migration of the antimicrobial nanoparticles to be effective. Such coatings can support reducing public health issues related to microbial cross-contamination in areas such as food processing, hospitals, and water purification.

Development of Engineered Bacteriophages for Escherichia coli Detection and High-Throughput Antibiotic Resistance Determination

T7 bacteriophages (phages) have been genetically engineered to carry the lacZ operon, enabling the overexpression of beta-galactosidase (β-gal) during phage infection and allowing for the enhanced colorimetric detection of Escherichia coli (E. coli). Following the phage infection of E. coli, the enzymatic activity of the released β-gal was monitored using a colorimetric substrate. Compared with a control T7 phage, our T7lacZ phage generated significantly higher levels of β-gal expression following phage infection, enabling a lower limit of detection for E. coli cells. Using this engineered T7lacZ phage, we were able to detect E. coli cells at 10 CFU·mL-1 within 7 h. Furthermore, we demonstrated the potential for phage-based sensing of bacteria antibiotic resistance profiling using our T7lacZ phage, and subsequent β-gal expression to detect antibiotic resistant profile of E. coli strains.

Lyophilized Engineered Phages for Escherichia coli Detection in Food Matrices

Ease of use, low cost, and convenient transport are the key requirements for a commercial bacteria detection kit designed for resource-limited settings. Here, we report the colorimetric detection of Escherichia coli (E. coli) in food samples using freeze-dried engineered bacteriophages (phages). In this approach, we have engineered T7 phages to carry the lacZ operon driven by T7 promoter to overexpress reporter enzymes. The engineered phages were freeze-dried in a water-soluble polymer for storage and transportation. When used for the detection of E. coli cells, the intracellular enzyme [β-galactosidase (β-gal)] was overexpressed and released into the surrounding media, providing an enzyme-amplified colorimetric signal. Using this strategy, we were able to detect E. coli cells at the concentration of 102 CFU mL-1 in food samples without the need for sophisticated instruments or skilled operators.

Facile hierarchical assembly of gold particle decorated conductive polymer nanofibers for electrochemical sensing

In this study, we successfully applied vapor-phase polymerization towards the synthesis of PEDOT nanofibers which were subsequently functionalized with gold particles and used as electrodes for electrochemical sensing. Two methods were used to synthesize the PEDOT nanofibers including (1) electrospinning followed by vapor-phase polymerization (EVP), and (2) one-step vapor-phase polymerization (OSVP). The average diameter of EVP fibers was approximately 350 nm, and OSVP was approximately 200 nm. Gold particles (∼500 nm) were synthesized by an oxidation-reduction reaction between gold precursors and residue EDOT monomers on the surface of the PEDOT nanofibers. In order to investigate the electrochemical performance of these electrodes, ascorbic acid was chosen as an analyte model. Our results indicated that PEDOT nanofiber electrodes showed an enhanced response with respect to bare gold electrodes. Furthermore, the OSVP PEDOT nanofibers with gold particles demonstrated the highest sensitivity at low ascorbic acid concentrations. These hierarchically assembled, gold particle-decorated, conductive polymer nanofibers were further fabricated into flexible electrodes, demonstrating a potential in advanced applications such as wearable electronics.