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Rapid detection of foodborne pathogens is essential to preventing foodborne illness outbreaks. Before detection can occur, however, it is often necessary to extract and concentrate bacteria. Conventional methods such as centrifugation, filtration, and immunomagnetic separation can often be time-consuming, ineffective, or costly when working with complex food matrices. This work used cost-effective glycan-coated magnetic nanoparticles (MNPs) for rapid concentration of Escherichia coli O157, Listeria monocytogenes, and Staphylococcus aureus. Glycan-coated MNPs were used to concentrate bacteria from both buffer solution and food matrices while examining the effect of factors including solution pH, bacterial concentration, and target bacterial species. In both pH 7 and reduced pH experiments, successful extraction of bacterial cells occurred in all food matrices and bacteria tested. In neutral pH buffer solution, bacteria were concentrated to 4.55 ± 1.17, 31.68 ± 6.10 and 64.27 ± 16.78 times their initial concentration (mean ± standard deviation) for E. coli, L. monocytogenes and S. aureus, respectively. Successful bacterial concentration occurred in several food matrices, including S. aureus in milk (pH 6), L. monocytogenes in sausage (pH 7), and E. coli O157 in flour (pH 7). The insights gained may facilitate future applications of glycan-coated MNPs to extract foodborne pathogens.

Carbapenem-resistant Enterobacterales (CRE) are one of the major global issues needing attention. Among them, carbapenemase-producing (CP) E. coli strains are commonly found in clinical and biological samples. Rapid and cost-effective detection of such strains is critical in minimizing their deleterious impact. While promising progress is being made in rapid detection platforms, separation and enrichment of bacteria are required to ensure the detection of low bacterial counts. The current separation methods, such as centrifugation, filtration, electrophoresis, and immunomagnetic separation, are often tedious, expensive, or ineffective for clinical and biological samples. Further, the extraction and concentration of antimicrobial-resistant bacteria (ARB) are not well documented. Thus, this study assessed the applicability of cost-effective glycan-coated magnetic nanoparticles (gMNPs) for simple and rapid extraction of CP E. coli. The study included two resistant (R)strains: Klebsiella pneumoniae carbapenemase (KPC)-producing E. coli (R: KPC) and New Delhi metallo-β-lactamase (NDM)-producing E. coli (R: NDM). A susceptible E. coli (S) strain was used as a control, a reference bacterium. The gMNPs successfully extracted and concentrated E. coli (R) and E. coli (S) at low concentrations from large volumes of buffer solution, water, and food samples. The gMNPs concentrated up to two and five times their initial concentration for E. coli (R) and E. coli (S) in the buffer solution, respectively. In water and food samples, the concentration of E. coli (S) and E. coli (R) were similar and ranged 1–3 times their initial inoculation. A variation in the concentration from different food samples was seen, displaying the impact of food microstructure and natural microflora. The cost-effective and rapid bacterial cell capture by gMNPs was achieved in 15 min, and its successful binding to the bacterial cells in the buffer solution and food matrices was also confirmed using Transmission Electron Microscopy (TEM). These results show promising applications of gMNPs to extract pathogens and ARB from biological samples.

One of the most prevalent causes of foodborne illnesses worldwide is staphylococcal food poisoning. This study aimed to provide a robust method to extract the bacteria Staphylococcus aureus from food samples using glycan-coated magnetic nanoparticles (MNPs). Then, a cost-effective multi-probe genomic biosensor was designed to detect the nuc gene of S. aureus rapidly in different food matrices. This biosensor utilized gold nanoparticles and two DNA oligonucleotide probes combined to produce a plasmonic/colorimetric response to inform users if the sample was positive for S. aureus. In addition, the specificity and sensitivity of the biosensor were determined. For the specificity trials, the S. aureus biosensor was compared with the extracted DNA of Escherichia coli, Salmonella enterica serovar Enteritidis (SE), and Bacillus cereus. The sensitivity tests showed that the biosensor could detect as low as 2.5 ng/µL of the target DNA with a linear range of up to 20 ng/µL of DNA. With further research, this simple and cost-effective biosensor can rapidly identify foodborne pathogens from large-volume samples.

Infectious disease outbreaks have caused thousands of deaths and hospitalizations, along with severe negative global economic impacts. Among these, infections caused by antimicrobial-resistant microorganisms are a major growing concern. The misuse and overuse of antimicrobials have resulted in the emergence of antimicrobial resistance (AMR) worldwide. Carbapenem-resistant Enterobacterales (CRE) are among the bacteria that need urgent attention globally. The emergence and spread of carbapenem-resistant bacteria are mainly due to the rapid dissemination of genes that encode carbapenemases through horizontal gene transfer (HGT). The rapid dissemination enables the development of host colonization and infection cases in humans who do not use the antibiotic (carbapenem) or those who are hospitalized but interacting with environments and hosts colonized with carbapenemase-producing (CP) bacteria. There are continuing efforts to characterize and differentiate carbapenem-resistant bacteria from susceptible bacteria to allow for the appropriate diagnosis, treatment, prevention, and control of infections. This review presents an overview of the factors that cause the emergence of AMR, particularly CRE, where they have been reported, and then, it outlines carbapenemases and how they are disseminated through humans, the environment, and food systems. Then, current and emerging techniques for the detection and surveillance of AMR, primarily CRE, and gaps in detection technologies are presented. This review can assist in developing prevention and control measures to minimize the spread of carbapenem resistance in the human ecosystem, including hospitals, food supply chains, and water treatment facilities. Furthermore, the development of rapid and affordable detection techniques is helpful in controlling the negative impact of infections caused by AMR/CRE. Since delays in diagnostics and appropriate antibiotic treatment for such infections lead to increased mortality rates and hospital costs, it is, therefore, imperative that rapid tests be a priority.

Antimicrobial resistance (AMR) is a global public health issue, and the rise of carbapenem-resistant bacteria needs attention. While progress is being made in the rapid detection of resistant bacteria, affordability and simplicity of detection still need to be addressed. This paper presents a nanoparticle-based plasmonic biosensor for detecting the carbapenemase-producing bacteria, particularly the beta-lactam Klebsiella pneumoniae carbapenemase (blaKPC) gene. The biosensor used dextrin-coated gold nanoparticles (GNPs) and an oligonucleotide probe specific to blaKPC to detect the target DNA in the sample within 30 min. The GNP-based plasmonic biosensor was tested in 47 bacterial isolates: 14 KPC-producing target bacteria and 33 non-target bacteria. The stability of GNPs, confirmed by the maintenance of their red appearance, indicated the presence of target DNA due to probe-binding and GNP protection. The absence of target DNA was indicated by the agglomeration of GNPs, corresponding to a color change from red to blue or purple. The plasmonic detection was quantified with absorbance spectra measurements. The biosensor successfully detected and differentiated the target from non-target samples with a detection limit of 2.5 ng/μL, equivalent to ~103 CFU/mL. The diagnostic sensitivity and specificity were found to be 79% and 97%, respectively. The GNP plasmonic biosensor is simple, rapid, and cost-effective in detecting blaKPC-positive bacteria.

Rapid detection of foodborne pathogens such as E. coli O157 is essential in reducing the prevalence of foodborne illness and subsequent complications. Due to their unique colorimetric properties, gold nanoparticles (GNPs) can be applied in biosensor development for affordability and accessibility. In this work, a GNP biosensor was designed for visual differentiation between target (E. coli O157:H7) and non-target DNA samples. Results of DNA extracted from pure cultures indicate high specificity and sensitivity to as little as 2.5 ng/µL E. coli O157 DNA. Further, the biosensor successfully identified DNA extracted from flour contaminated with E. coli O157, with no false positives for flour contaminated with non-target bacteria. After genomic extraction, this assay can be performed in as little as 30 min. In addition, food sample testing was successful at detecting approximately 103 CFU/mL of E. coli O157 magnetically extracted from flour after only a 4 h incubation step. As a proof of concept, these results demonstrate the capabilities of this GNP biosensor for low-cost and rapid foodborne pathogen detection.

Despite its reduced sensitivity, sputum smear microscopy (SSM) remains the main diagnostic test for detecting tuberculosis in many parts of the world. A new diagnostic technique, the magnetic nanoparticle-based colorimetric biosensing assay (NCBA) was optimized by evaluating different concentrations of glycan-functionalized magnetic nanoparticles (GMNP) and Tween 80 to improve the acid-fast bacilli (AFB) count. Comparative analysis was performed on 225 sputum smears: 30 with SSM, 107 with NCBA at different GMNP concentrations, and 88 with NCBA-Tween 80 at various concentrations and incubation times. AFB quantification was performed by adding the total number of AFB in all fields per smear and classified according to standard guidelines (scanty, 1+, 2+ and 3+). Smears by NCBA with low GMNP concentrations (≤1.5 mg/mL) showed higher AFB quantification compared to SSM. Cell enrichment of sputum samples by combining NCBA-GMNP, incubated with Tween 80 (5%) for three minutes, improved capture efficiency and increased AFB detection up to 445% over SSM. NCBA with Tween 80 offers the opportunity to improve TB diagnostics, mainly in paucibacillary cases. As this method provides biosafety with a simple and inexpensive methodology that obtains results in a short time, it might be considered as a point-of-care TB diagnostic method in regions where resources are limited.

Abstract Background Nuclear magnetic resonance (NMR) technique is a powerful analytical tool in determining the presence of bacterial contaminants in complex biological samples. In this paper, a portable NMR-based (pNMR) biosensor and assay to detect the foodborne bacteria Escherichia coli O157:H7 is reported. It uses antibody-functionalized polymer-coated magnetic nanoparticles as proximity biomarker of the bacteria which accelerates NMR resonance signal decay. Results The pNMR biosensor operates at 0.47 Tesla of magnetic strength and consists of a high-power pulsed RF transmitter and an ultra-low noise sensing circuitry capable of detecting weak NMR signal at 0.1 μV. The pNMR biosensor assay and sensing mechanism is used in detecting E. coli O157:H7 bacteria in drinking water and milk samples. Experimental results demonstrate that by adding a filtration step in the assay, the pNMR biosensor is able to detect E. coli O157:H7 as low as 76 CFU/mL in water samples and as low as 92 CFU/mL in milk samples in about one min. Conclusion The pNMR biosensor assay and sensing system is innovative for foodborne bacterial detection in food matrices. The lowest detection level for E. coli O157:H7 in water and milk samples is essentially 101 CFU/mL. Although the linear range of detection is only from 101 to 104 CFU/mL, the wider detection range spans from 101 CFU/mL to 107 CFU/mL. Existing pNMR biosensors have detection limits at 103-104 CFU/mL only. The detection technique can be extended to other microbial or viral organisms by merely changing the specificity of the antibodies. Besides food safety, the pNMR biosensor described in this paper has potential to be applied as a rapid detection device in biodefense and healthcare diagnostic applications.

In this manuscript, we discuss relevant socioeconomic factors for developing and implementing sensor analytic point solutions (SNAPS) as point-of-care tools to serve impoverished communities. The distinct economic, environmental, cultural, and ethical paradigms that affect economically disadvantaged users add complexity to the process of technology development and deployment beyond the science and engineering issues. We begin by contextualizing the environmental burden of disease in select low-income regions around the world, including environmental hazards at work, home, and the broader community environment, where SNAPS may be helpful in the prevention and mitigation of human exposure to harmful biological vectors and chemical agents. We offer examples of SNAPS designed for economically disadvantaged users, specifically for supporting decision-making in cases of tuberculosis (TB) infection and mercury exposure. We follow-up by discussing the economic challenges that are involved in the phased implementation of diagnostic tools in low-income markets and describe a micropayment-based systems-as-a-service approach (pay-a-penny-per-use—PAPPU), which may be catalytic for the adoption of low-end, low-margin, low-research, and the development SNAPS. Finally, we provide some insights into the social and ethical considerations for the assimilation of SNAPS to improve health outcomes in marginalized communities.

Food defense requires the means to efficiently screen large volumes of food for microbial pathogens. Even rapid detection methods often require lengthy enrichment steps, making them impractical for this application. There is a great need for rapid, sensitive, specific, and inexpensive methods for extracting and concentrating microbial pathogens from food. In this study, an immuno-magnetic separation (IMS) methodology was developed for Escherichia coli O157:H7, using electrically active magnetic nanoparticles (EAMNPs). The analytical specificity of the IMS method was evaluated against Escherichia coli O55:H7 and Shigella boydii, and was improved over previous protocols by the addition of sodium chloride during the conjugation of antibodies onto MNPs. The analytical sensitivity of the IMS method was greatest when a high concentration of antibodies (1.0 mg/mL) was present during conjugation. EAMNP concentrations of 1.0 and 0.5 mg/mL provided optimal analytical sensitivity and analytical specificity. The entire IMS procedure requires only 35 min, and antibody-conjugated MNPs show no decline in performance up to 149 days after conjugation. This analytically sensitive and specific extraction protocol has excellent longevity and shows promise as an effective extraction for multiple electrochemical biosensor applications.

Dengue is a rapidly spreading mosquito-borne viral disease. Early diagnosis is important for clinical screening, medical management, and disease surveillance. The objective of this study was to develop a colorimetric lateral flow biosensor (LFB) for the visual detection of dengue-1 RNA using dextrin-capped gold nanoparticle (AuNP) as label. The detection was based on nucleic acid sandwich-type hybridization among AuNP-labeled DNA reporter probe, dengue-1 target RNA, and dengue-1 specific DNA capture probe immobilized on the nitrocellulose membrane. Positive test generated a red test line on the LFB strip, which enabled visual detection. The optimized biosensor has a cut-off value of 0.01 µM using synthetic dengue-1 target. Proof-of-concept application of the biosensor detected dengue-1 virus in pooled human sera with a cut-off value of 1.2 × 104 pfu/mL. The extracted viral RNA, when coupled with nucleic acid sequence-based amplification (NASBA), was detected on the LFB in 20 min. This study first demonstrates the applicability of dextrin-capped AuNP as label for lateral flow assay. The biosensor being developed provides a promising diagnostic platform for early detection of dengue infection in high-risk resource-limited areas.

Development of new diagnostic platforms that incorporate lab-on-a-chip technologies for portable assays is driving the need for rapid, simple, low cost methods to prepare samples for downstream processing or detection. An important component of the sample preparation process is cell lysis. In this work, a simple microfluidic thermal lysis device is used to quickly release intracellular nucleic acids and proteins without the need for additional reagents or beads used in traditional chemical or mechanical methods (e.g., chaotropic salts or bead beating). On-chip lysis is demonstrated in a multi-turn serpentine microchannel with external temperature control via an attached resistive heater. Lysis was confirmed for Escherichia coli by fluorescent viability assay, release of ATP measured with bioluminescent assay, release of DNA measured by fluorometry and qPCR, as well as bacterial culture. Results comparable to standard lysis techniques were achievable at temperatures greater than 65 °C and heating durations between 1 and 60 s.

Although real-time PCR (RT-PCR) has become a diagnostic standard for rapid identification of bacterial species, typical methods remain time-intensive due to sample preparation and amplification cycle times. The assay described in this work incorporates on-chip dielectrophoretic capture and concentration of bacterial cells, thermal lysis, cell permeabilization, and nucleic acid denaturation and fluorescence resonance energy transfer assisted in situ hybridization (FRET-ISH) species identification. Combining these techniques leverages the benefits of all of them, allowing identification to be accomplished completely on chip less than thirty minutes after receipt of sample, compared to multiple hours required by traditional RT-PCR and its requisite sample preparation.

Bacterial pathogens pose an increasing food safety and bioterrorism concern. Current DNA detection methods utilizing sensitive nanotechnology and biosensors have shown excellent detection, but require expensive and time-consuming polymerase chain reaction (PCR) to amplify DNA targets; thus, a faster, more economical method is still essential. In this proof-of-concept study, we investigated the ability of a gold nanoparticle-DNA (AuNP-DNA) biosensor to detect non-PCR amplified genomic Salmonella enterica serovar Enteritidis (S. enteritidis) DNA, from pure or mixed bacterial culture and spiked liquid matrices. Non-PCR amplified DNA was hybridized into sandwich-like structures (magnetic nanoparticles/DNA/AuNPs) and analyzed through detection of gold voltammetric peaks using differential pulse voltammetry. Our preliminary data indicate that non-PCR amplified genomic DNA can be detected at a concentration as low as 100 ng/mL from bacterial cultures and spiked liquid matrices, similar to reported PCR amplified detection levels. These findings also suggest that AuNP-DNA biosensors are a first step towards a viable detection method of bacterial pathogens, in particular, for resource-limited settings, such as field-based or economically limited conditions. Future efforts will focus on further optimization of the DNA extraction method and AuNP-biosensors, to increase sensitivity at lower DNA target concentrations from food matrices comparable to PCR amplified DNA detection strategies.

Biological defense and security applications demand rapid, sensitive detection of bacterial pathogens. This work presents a novel qualitative electrochemical detection technique which is applied to two representative bacterial pathogens, Bacillus cereus (as a surrogate for B. anthracis) and Escherichia coli O157:H7, resulting in detection limits of 40 CFU/mL and 6 CFU/mL, respectively, from pure culture. Cyclic voltammetry is combined with immunomagnetic separation in a rapid method requiring approximately 1 h for presumptive positive/negative results. An immunofunctionalized magnetic/polyaniline core/shell nano-particle (c/sNP) is employed to extract target cells from the sample solution and magnetically position them on a screen-printed carbon electrode (SPCE) sensor. The presence of target cells significantly inhibits current flow between the electrically active c/sNPs and SPCE. This method has the potential to be adapted for a wide variety of target organisms and sample matrices, and to become a fully portable system for routine monitoring or emergency detection of bacterial pathogens.

Access to community-based point-of-care, low-cost, and sensitive tuberculosis (TB) diagnostics remains an unmet need. Objective: The objective of this study was to combine principles in nanotechnology, TB biology, glycochemistry, and engineering, for the development of a nanoparticle-based colorimetric biosensing assay (NCBA) to quickly and inexpensively detect acid-fast bacilli (AFB) in sputum samples. Methods: In NCBA, the isolation of AFB from sputum samples was accomplished through glycan-coated magnetic nanoparticles (GMNP) interacting with AFB and then using a simple magnet to separate the GMNP-AFB complex. Acid-fastness and cording properties of mycobacteria were utilized to provide visually observable red-stained clumps of bacteria that were surrounded by brown nanoparticles under a light microscope on prepared smears. The NCBA technique was compared against sputum smear microscopy (SSM) and Xpert MTB/RIF in 500 samples from patients that were suspected to have TB. Results: Statistical analysis showed that NCBA had sensitivity and specificity performances in perfect agreement with Xpert MTB/RIF as gold standard for all 500 samples. SSM had a sensitivity of 40% for the same samples. Conclusion: NCBA technique yielded full agreement in terms of sensitivity and specificity with the Xpert MTB/RIF in 500 samples. The method is completed in 10–20 min through a simple process at an estimated cost of $0.10 per test. Implementation of NCBA in rural communities would help to increase case finding and case notification, and would support programs against drug-resistance. Its use at the first point-of-contact by patients in the healthcare system would facilitate quick treatment in a single clinical encounter, thus supporting the global “End TB Strategy” by 2035.

A new method using a magnetic nanoparticle-based colorimetric biosensing assay (NCBA) was compared with sputum smear microscopy (SSM) for the detection of pulmonary tuberculosis (PTB) in sputum samples. Studies were made to compare the NCBA against SSM using sputum samples collected from PTB patients prior to receiving treatment. Experiments were also conducted to determine the appropriate concentration of glycan-functionalized magnetic nanoparticles (GMNP) used in the NCBA and to evaluate the optimal digestion/decontamination solution to increase the extraction, concentration and detection of acid-fast bacilli (AFB). The optimized NCBA consisted of a 1:1 mixture of 0.4% NaOH and 4% N-acetyl-L-cysteine (NALC) to homogenize the sputum sample. Additionally, 10 mg/mL of GMNP was added to isolate and concentrate the AFB. All TB positive sputum samples were identified with an increased AFB count of 47% compared to SSM, demonstrating GMNP’s ability to extract and concentrate AFB. Results showed that NCBA increased AFB count compared to SSM, improving the grade from “1+„ (in SSM) to “2+„. Extending the finding to paucibacillary cases, there is the likelihood of a “scant„ grade to become “1+„. The assay uses a simple magnet and only costs $0.10/test. NCBA has great potential application in TB control programs.

The technique of molecular imprinting produces artificial receptor sites in apolymer that can be used in a biomimetic sensor. This research extends previous studies ofa molecularly imprinted polymer (MIP) biomimetic sensor for the small drug theophylline.The presence of theophylline in the biomimetic sensor was monitored by analyzing thepeak currents from cyclic voltammetry experiments. The functional working range of theMIP modified electrode was 2 - 4 mM theophylline. The concentration of theophyllinethat resulted in the best signal was 3 mM. The MIP sensor showed no response to thestructurally related molecule caffeine, and therefore was selective to the target analytetheophylline. This research will provide the foundation for future studies that will result indurable biomimetic sensors that can offer a viable alternative to current sensors.

In this study, a novel indium tin oxide (ITO)-polyaniline (Pani) biosensor wasdesigned, fabricated, and characterized. Initial testing was conducted for the detection ofbovine viral diarrhea virus (BVDV). The biosensor design was based upon the specific natureof antibodies to capture the target virus, and the conductive properties of self-doped Pani totranslate the antibody-antigen binding into a quantifying signal. The first part of the study wasto assess the feasibility of the self-doped Pani to be incorporated into the biosensor design byevaluating its several parameters, such as conductivity, physical structure, thermogravimetricproperties, and antibody-binding properties. The second part of the paper highlights thefabrication of the ITO-Pani biosensor to detect the presence of bovine viral diarrhea virus(BVDV) in pure culture. Although only BVDV culture was tested in this study, the biosensoris versatile for the detection of other pathogen of interest by changing the specificity of theantibodies.