Title: Utilizing HRPzyme, a cost-effective Vibrio parahaemolyticus detection method
Vibrio parahaemolyticus is a prominent infectious bacterium responsible for causing widespread cases of acute gastroenteritis in humans globally. In this regard, Colorimetric detection can be essentially used as a sensitive, rapid, and cost-effective detection method. In our research, we have developed a PCR-based detection platform integrated with HRPzyme and utilizing DNAzyme as a signaling probe which mimics peroxidase activity. The colorimetric signal is detectable at concentrations as low as 101 cfu mL−1 when measured with a spectrophotometer and at 103 cfu mL−1 through visual inspection. Additionally, extending the polyadenine length to 10 nucleotides resulted in a significant reduction in the background signaling of HRPzyme activity, yielding a relative intensity of 3.07 ± 0.23 arbitrary units (a.u.). Notably, even after a 120-min incubation period, there were no further changes observed in the colorimetric signal in positive samples, maintaining a consistent relative intensity of OD 410 = 0.55 ± 0.08. more »« less
This study validates a fiber optics-based Surface Enhanced Raman Spectroscopy (SERS) sensor for detecting Salmonella in raw turkey samples. The sensor uses nanoantenna arrays on a side-polished optical fiber core with a fixed periodicity of 0.77 µm to maximize the SERS signal intensity. A 3D-printed chamber and microstructure optimize light reflection, improving sensitivity. The sensor detects Salmonella at 0.4-0.5 CFU/ml in 10 minutes, offering cost-effective, portable pathogen detection.
Colorimetric enzyme-linked immunosorbent assay (ELISA) has been widely applied as the gold-standard method for cytokine detection for decades. However, it has become a critical challenge to further improve the detection sensitivity of ELISA, as it is limited by the catalytic activity of enzymes. Herein, we report an enhanced colorimetric ELISA for ultrasensitive detection of interleukin-6 (IL-6, as a model cytokine for demonstration) using Pd@Pt core@shell nanodendrites (Pd@Pt NDs) as peroxidase nanomimics (named “Pd@Pt ND ELISA”), pushing the sensitivity up to femtomolar level. Specifically, the Pd@Pt NDs are rationally engineered by depositing Pt atoms on Pd nanocubes (NCs) to generate rough dendrite-like Pt skins on the Pd surfaces via Volmer–Weber growth mode. They can be produced on a large scale with highly uniform size, shape, composition, and structure. They exhibit significantly enhanced peroxidase-like catalytic activity with catalytic constants (Kcat) more than 2000-fold higher than those of horseradish peroxidase (HRP, an enzyme commonly used in ELISA). Using Pd@Pt NDs as the signal labels, the Pd@Pt ND ELISA presents strong colorimetric signals for the quantitative determination of IL-6 with a wide dynamic range of 0.05–100 pg mL−1 and an ultralow detection limit of 0.044 pg mL−1 (1.7 fM). This detection limit is 21-fold lower than that of conventional HRP-based ELISA. The reproducibility and specificity of the Pd@Pt ND ELISA are excellent. More significantly, the Pd@Pt ND ELISA was validated for analyzing IL-6 in human serum samples with high accuracy and reliability through recovery tests. Our results demonstrate that the colorimetric Pd@Pt ND ELISA is a promising biosensing tool for ultrasensitive determination of cytokines and thus is expected to be applied in a variety of clinical diagnoses and fundamental biomedical studies.
Wang, Danhui; Hinkley, Troy; Chen, Juhong; Talbert, Joey N.; Nugen, Sam R.
(, The Analyst)
The monitoring of drinking water for indicators of fecal contamination is crucial for ensuring a safe supply. In this study, a novel electrochemical method was developed for the rapid and sensitive detection of Escherichia coli ( E. coli ) in drinking water. This strategy is based on the use of engineered bacteriophages (phages) to separate and concentrate target E. coli when conjugated with magnetic beads, and to facilitate the detection by expressing gold binding peptides fused alkaline phosphatase (GBPs-ALP). The fusion protein GBPs-ALP has both the enzymatic activity and the ability to directly bind onto a gold surface. This binding-peptide mediated immobilization method provided a novel and simple approach to immobilize proteins on a solid surface, requiring no post-translational modifications. The concentration of E. coli was determined by measuring the activity of the ALP on gold electrodes electrochemically using linear sweep voltammetry (LSV). This approach was successfully applied in the detection of E. coli in drinking water. We were able to detect 10 5 CFU mL −1 of E. coli within 4 hours. After 9 hours of preincubation, 1 CFU of E. coli in 100 mL of drinking water was detected with a total assay time of 12 hours. This approach compares favorably to the current EPA method and has the potential to be applied to detect different bacteria in other food matrices.
Wenbap, Pattarapong; Rattanarojpong, Triwit; Khunrae, Pongsak; Luangtongkum, Taradon; Erickson, Larry E.; Hansen, Ryan R.; Tuitemwong, Pravate
(, Journal of Nanomaterials)
Parvinzadeh Gashti, Mazeyar
(Ed.)
The simple, accurate, and rapid detection of foodborne pathogens is essential for public health. Development of an immunomagnetic separation (IMS) multiplex touchdown PCR (IMS–multiplex TD–PCR) assay for simultaneous detection and distinguishing of C. jejuni and C. coli is reported herein. Polyclonal antibody (pAb) against multiepitope antigen (MEA) was conjugated to ferromagnetic nanoparticles (FMNs) to produce anti-MEA FMNs. Optimal anti-MEA FMNs loading yielded 26.7 μg of immunoglobulin G (IgG) molecules per mg of FMNs with an average size of 72 ± 9 nm, corresponding to an 83% rate of pAb conjugation. Anti-MEA FMNs (20 μg) for IMS captured culturable C. jejuni cells at 3.54 × 10 2 colony-forming unit (CFU)/mL in pure culture, while higher amounts (40 and 60 μg) reduced the recovery. The scanning electron microscope (SEM) analysis revealed the attachment of anti-MEA FMNs to target bacteria, forming aggregated cells and magnetic nanoparticles in ellipse-like shapes. The subsequent multiplex TD–PCR assay simultaneously detected and distinguished C. jejuni and C. coli at 104 CFU/mL in mixed culture and at 103 CFU/mL for each individual species. Furthermore, the limit of detection (LOD) of the IMS–multiplex TD–PCR assay was 104 CFU/g in spiked chicken breast samples. Specificity was 100% for both C. jejuni and C. coli as none of the amplicons were detected in control samples where Campylobacter was absent. This assay is able to detect and distinguish C. jejuni and C. coli simultaneously and is simple, accurate, and rapid with a time to result of 4 h without an enrichment step, making it a promising approach for rapid and culture-free detection of Campylobacter in chicken products.
Almalaysha, Mohammed; Allen, Keara; Muhsin, Sura Abd-Alzahra; Bashir_Taas, Kamran; Carlson, Anna; Morey, Amit; Trout, Kate E; Zhang, Shuping; Almasri, Mahmoud F
(, SPIE)
Rapp, Bastian E; Dalton, Colin
(Ed.)
This study presents the development of a highly sensitive microfluidic-based impedance biosensor designed for rapid detection and identification of Salmonella Infantis in raw turkey samples, with a limit of detection (LOD) as low as 1 CFU/ml in 70 minutes detection time. The biosensor is equipped with novel focusing and trapping regions, significantly enhancing its sensitivity by concentrating and trapping Salmonella cells in the detection region. Salmonella cells labeled with fluorescent dyes were used to validate the functionality of the focusing and trapping mechanism, confirming the biosensor's ability to concentrate and trap Salmonella cells.
@article{osti_10513831,
place = {Country unknown/Code not available},
title = {Utilizing HRPzyme, a cost-effective Vibrio parahaemolyticus detection method},
url = {https://par.nsf.gov/biblio/10513831},
DOI = {10.1016/j.lwt.2023.115461},
abstractNote = {Vibrio parahaemolyticus is a prominent infectious bacterium responsible for causing widespread cases of acute gastroenteritis in humans globally. In this regard, Colorimetric detection can be essentially used as a sensitive, rapid, and cost-effective detection method. In our research, we have developed a PCR-based detection platform integrated with HRPzyme and utilizing DNAzyme as a signaling probe which mimics peroxidase activity. The colorimetric signal is detectable at concentrations as low as 101 cfu mL−1 when measured with a spectrophotometer and at 103 cfu mL−1 through visual inspection. Additionally, extending the polyadenine length to 10 nucleotides resulted in a significant reduction in the background signaling of HRPzyme activity, yielding a relative intensity of 3.07 ± 0.23 arbitrary units (a.u.). Notably, even after a 120-min incubation period, there were no further changes observed in the colorimetric signal in positive samples, maintaining a consistent relative intensity of OD 410 = 0.55 ± 0.08.},
journal = {LWT},
volume = {189},
number = {C},
publisher = {Elsevier},
author = {Parsaeimehr, Ali and Ozbay, Gulnihal},
}
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