A novel aggregation-induced emission probe-linked phage sorbent assay for virulent bacteria strain imaging and on-site detection
Graphical abstract
Introduction
Food pathogen bacteria can pose serious harm to human beings [[1], [2], [3]]. At the same time, there are strong and weak strains of the same kind of bacteria. The virulent strain will do great harm even at low concentrations, while the attenuated strain will not cause serious harm even at very high concentrations [4,5]. Therefore, it is important to discriminate between the virulent and attenuated strains of the same pathogen bacteria. Furthermore, on-site detection and imaging of virulent food-borne pathogens and their virulent strains is highly important to human health and life.
The plate counting method is the gold standard to detect pathogen bacteria, however, its manipulation is tedious and normally takes 2–7 days for proliferation then for biochemical analysis [[6], [7], [8]]. Nuclear amplification technology, e.g., Polymerase Chain Reaction (PCR), Loop-Mediated Isothermal Amplification (LAMP), photoelectrochemical (PEC) can discriminate different strains by amplifying and recognizing the specific gene sequences of different strains [[9], [10], [11], [12]]. However, it is not easy to be on-site operated because of the aerosol pollution and negative pressure environment [13]. The biosensors based on antibody and aptamer as recognition components can be employed for on-site and rapid detection of food pathogen bacteria [14,15]. However, it can also hardly discriminate the strain. Phages are viruses that attack and eat bacteria. They can not only recognize different strains of one bacteria but also more readily combine with the bacteria [16]. Therefore, the phage was regarded as an ideal candidate biological recognition component for fabricating the biosensor probe, which can discriminate virulent strains in the pathogen [17]. Up to now, there have been some studies about phage-based biosensors for detecting some virulent strains of food pathogens. Shabani et al. [18] developed an electrochemical sensor based on T4 Phage that was used to detect the host bacterial E. coli, and the detection limit was 104 CFU mL−1. Zhou et al. [19] used electrochemical impedance spectroscopy (EIS) to monitor E. coli by T2 phage which was fixed on the CNT-modified electrode, and the detection limit was 103 CFU mL−1. But electrochemical sensors have some drawbacks, such as the possibility of some electrode surface desorption, and the sensitivity of the phage surface uniformity has certain requirements [20,21]. Tang et al. [22] successfully imaged Pseudomonas aeruginosa and killed it specifically by photodynamic therapy by combining phage with an AIE probe to form a bio-composite probe. All these studies have demonstrated that phages are good candidates for bacterial detection and have strong specificity. Therefore, we hoped to develop a phage-based assay for detecting and imaging E. coli O157:H7 which is the toxic strain of E. coli [23]. Moreover, to construct a phage-based assay for on-site detection, it is necessary to develop some suitable sensing interfaces and signal tags to convert the amount of the target bacteria to a certain signal. The 96-well plate is an ideal platform for embedding antibodies and other proteins as recognition components. Based on it, some on-site detection assays such as enzyme-linked immunosorbent assay (ELISA) were developed and convenient for detection through the enzyme reader [24]. The fluorescent-based imaging and detection assay are more suitable for visual measurement.
For bacteria imaging, fluorescence quenching of the labeling dye is one major issue encountered in fluorescence microscopy and confocal laser microscopy [25]. Because the laser scanning confocal microscope has stronger power and more accurate beam focusing, the photo-bleaching effect of specimens is more obvious than that of an ordinary fluorescence microscope, and the fluorescence of dyes can gradually decrease or disappear during continuous observation. Furthermore, many conventional fluorescent probes, such as N,N-dicyclohexyl-1,7-dibromo-3,4,9,10-perylenetetracarboxylic diimide (DDPA), are fluorescently quenched at high concentrations or aggregated state, which is the notorious aggregation caused quenching (ACQ), and some probes have ACQ effects even in a diluted state [26]. Compared with them, the AIE luminogens (AIEgens) exhibit more intensive signal and anti-photobleaching features [27,28]. Its high signal-to-noise ratio may open a new way for imaging or detecting bacteria [29]. Normally, AIEgens for detecting bacteria require identification components such as phenylboronic acid and quaternary ammonium [[30], [31], [32]]. Tang group developed a series of TPE-based AIEgens containing boronic acid group for bacterial detection [32]. The phenylboronic acid group on the AIEgens can covalently combine with 1,2-diol or 1,3-diol of peptidoglycan on the bacterial surface [33,34]. Then, the AIEgens would aggregate to prevent the rotation of the C–C bond between benzene and alkene groups in the molecular, and emit high fluorescence light. However, AIEgens containing the boronic acid group cannot specifically recognize bacteria strains. Moreover, according to the reports of Tang et al. [32], the synthesis of TPE with a boronic acid group normally required the use of n-butyl lithium as a catalyst. It is flammable, explosive, and dangerous for reacting at −78 °C. All these setbacks limit the application of TPE with the boric acid group's bacterial detection. Therefore, in this study, we hoped to design a simple and safe method to efficiently synthesize the AIE for bacterial detection and AIE imaging. Like ELISA, if the antibody was replaced by the phage to immobilize on the 96-well plates and the enzyme was replaced by the AIE probe, a novel AIE probe-linked phage sorbent assay (ALPSA) can be developed. It can realize the specific fluorescent imaging and analysis of virulent strains.
Herein, a novel AIE dye, TPE containing a phenylboronic acid (TPE-PBA), was firstly facile synthesized for imaging and detecting bacteria using the Suzuki coupling reaction. Then, an AIE probe-linked phage sorbent assay (ALPSA) was developed with TPE-PBA as the signal tag. It was successfully developed for imaging and detecting E. coli O157:H7. We also use PDDA to connect the phage at the bottom of the 96-well plate for orientation and enhance the phages’ binding capacity to E. coli O157:H7. The assay is simple to operate and suitable for on-site analysis of the virulent strain-E. coli O157:H7. The preparation of the AIEgen, imaging, and detection of E. coli O157:H7 was depicted in Scheme 1.
Section snippets
Reagents, materials, and instrumentation
See Supplementary Material.
AIE imaging for bacteria by centrifugation and resuspension
The fresh bacteria (bacteria culture was in Supplementary Material) were packed into 4 tubes, then the culture medium was removed by centrifugation (5000 rpm, 5 min). The resuspension of 1.0 mL of 0.1 M Tris-HCl buffer (pH 8.5) was replaced, and so on three times. In the final resuspension, 1.0 mL of mixed solution (Tris-HCl: DMSO = 9:1) was added into bacteria for resuspending, and TPE-PBA (4'-(1,2,2-triphenylvinyl)-[1,1′-biphenyl]-4-yl)boronic acid) was dissolved in
The characterization of TPE-PBA AIEgen
In the study, TPE-OBO (4,4,5,5-tetramethyl-2-(4-(1,2,2-triphenylvinyl)phenyl)-1,3,2-dioxaborolane) and TPE-PBA was synthesized (the detailed synthetic procedure was described in Supplementary Material) and characterized by 1H NMR,13C NMR, and high-resolution mass spectra (HR-MS), which were showed in Fig. S1-Fig. S6. To characterize the optical properties of TPE-PBA, the ultraviolet (UV) absorption spectrum of TPE-PBA was done and shown in Fig. 1a. There were two absorption peaks at about
Conclusion
In conclusion, a novel AIEgen named TPE-PBA was simply synthesized and employed as the imaging dye for bacteria. Based on TPE-PBA as signal tag, a novel AIEgen-linked phage sorbent assay (ALPSA) for virulent bacteria strain imaging and on-site detection strain E. coli O157:H7. 96-well plate was employed as the detection platform and POCT. The synthesis of TPE-PBA was simple, and the assay was low-cost. And more importantly, the assay exhibited high sensitivity and specificity toward E. coli
CRediT authorship contribution statement
Jiahui Teng: Formal analysis, Validation, Investigation, Data curation, Visualization, Methodology, Software, Conceptualization, Writing – original draft, preparation. Cong Cao: Data curation, Visualization, Validation, Investigation, Writing – review & editing. Zhenzhong Yu: Data curation, Validation, Investigation. Hongzhen Xie: Data curation, Investigation. Fubang Liu: Data curation. Chong Xu: Data curation. Yuting Cao: Data curation, Writing – review & editing, Validation, Investigation.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgment
This work was supported by the National Natural Science Foundation of China (No. 21974074), Zhejiang Province Public Welfare Technology Application Research Analysis Test Plan (No. LGC20B050006, LG20B050004), Major scientific and technological tasks of Ningbo (Grant no. 2022Z170, 2022S011), the Introduced Talents Scientific Research Start-up Foundation of Ningbo University (No. KJRC202110012), and K. C. Wong Magna Fund in Ningbo University of China.
References (36)
- et al.
Foodborne pathogens and their toxins
J. Proteonomics
(2016) - et al.
Correlation between clinico-pathological outcome and typing of Haemophilus parasuis field strains
Vet. Microbiol.
(2010) Exposing serum susceptibility in Haemophilus parasuis
Vet. J.
(2013)- et al.
Sequencing and analysis of the Escherichia coli serogroup O117, O126, and O146 O-antigen gene clusters and development of PCR assays targeting serogroup O117-, O126-, and O146-specific DNA sequences
Mol. Cell. Probes
(2007) - et al.
A highly specific and sensitive loop-mediated isothermal amplification method for the detection of Escherichia coli O157:H7
Microb. Pathog.
(2016) - et al.
Bio-bar-code-based photoelectrochemical immunoassay for sensitive detection of prostate-specific antigen using rolling circle amplification and enzymatic biocatalytic precipitation
Biosens. Bioelectron.
(2018) - et al.
CoOOH nanosheets-coated g-C3N4/CuInS2 nanohybrids for photoelectrochemical biosensor of carcinoembryonic antigen coupling hybridization chain reaction with etching reaction
Sens. Actuators, B
(2020) - et al.
Chicken egg yolk antibody (IgY) as diagnostics and therapeutics in parasitic infections - a review
Int. J. Biol. Macromol.
(2019) - et al.
An aptamer cocktail-functionalized photocatalyst with enhanced antibacterial efficiency towards target bacteria
J. Hazard Mater.
(2016) - et al.
ArgO 145, a Stx2a prophage of a bovine O145:H-STEC strain, is closely related to phages of virulent human strains
Infect. Genet. Evol.
(2018)