Detection Of Bacterial Spores Research Articles

Colorimetric aptasensor for detection of Bacillus cytotoxicus spores in milk and ready-to-use food

The high incidence of foodborne diseases caused by pathogenic bacteria raises concerns worldwide and imposes considerable public healthcare challenges. This is especially observed with dormant spores of Bacilli, which can often survive treatments used by the food industry to kill growing bacteria. The early and rapid detection of bacterial spores is essential to ensure food safety. Commercial availability of such a test will present a high potential for food sector. We present a point-of-need colorimetric assay for detection of Bacillus cytotoxicus spores in food. The detection principle is based on spore-enhanced peroxidase-like catalytic activity of gold nanoparticles. The sensing platform consists of a microtube containing gold nanoparticles (AuNPs), and magnetic particles (MPs), both conjugated with specific aptamer BAS6R that recognize B. cytotoxicus spores. Upon the addition of the sample, spores were determined as present by the enhanced color change of the solution, due to the oxidation of tetramethylbenidine (TMB) with H2O2. The assay was evaluated by the naked eye (on/off) and quantitatively with use of a spectrophotometer. BAS6R@AuNPs aptasensor coupled to BAS6R@MPs proved to be highly sensitive, achieving the naked-eye limit of detection as low as 102 cfu/mL in water and milk, and 104 cfu/mL in mashed potatoes. Moreover, discrimination between spores of B. cytotoxicus and B. subtilis as well as bacterial vegetative cells was achieved in contaminated food samples, providing a good selectivity. This work provides a promising proof of concept for the development of instrument-free, low-cost and rapid assay for Bacillus cytotoxicus spore detection, which is able to compete in sensitivity with conventional costly and time-consuming laboratory analyses.

Single-shot detection of bacterial spores with Yb-laser-based CARS spectroscopy.

We present a system based on a high-energy femtosecond ytterbium laser seeding an optical parametric amplifier and a photonic crystal hollow core fiber (PCHCF) compressor for coherent anti-Stokes Raman scattering (CARS) spectroscopy. The PCHCF provides spectral broadening of the Stokes pulse which is then compressed to a duration matched to that of the pump pulse. In these conditions, the excitation efficiency of vibrational levels in the target molecules is largely improved, as the time gating effect due to the mismatch between the durations of the pump and Stokes pulses is avoided. Experiments are presented along with a theoretical model to quantify expected improvement of sensitivity. The system is used to detect bacterial spores deposited on a surface with a single laser shot at unprecedented signal-to-noise ratio.

Standoff detection of bacterial spores by field deployable coherent Raman spectroscopy

Vibrational spectroscopies offer great potential for standoff detection of chemical and biological warfare agents, avoiding contamination to the operator and equipment. Among them, particularly promising is Coherent anti-Stokes Raman scattering (CARS) spectroscopy, using synchronized pump/Stokes laser pulses to set up a vibrational coherence of target molecules at a laser focus, which is read by further interaction with a probe pulse, resulting in the emission of a coherent beam detectable at a distance. CARS has previously demonstrated the capability to detect bacterial spores based on the Raman spectrum of the characteristic molecule calcium dipicolinate (CaDPA); however, a complex and bulky laser technology, which is only suitable for a laboratory environment, was employed. Here we develop a broadband CARS setup based on a compact, industrial grade ytterbium laser system. We demonstrate high signal-to-noise ratio detection of Bacillus atrophaeus spores at a concentration of 105 cfu/mm2, at a standoff distance of 1 m, and an acquisition time of 1 s. Our system, which combines chemical specificity and sensitivity along with improved ruggedness and portability, paves the way to a new generation of instruments for real-world standoff detection of chemical and biological threats.

Fluorophore molecule loaded in Tb-MOF for dual-channel fluorescence chemosensor for consecutive visual detection of bacterial spores and dichromate anion

Developing multifunctional luminescent switches for detecting various poisons are urgent given the continuous deterioration of the environment. Herein, a new porous fluorescent metal–organic framework [Tb2(TPTC)(DMA)4(NO3)2]n(HPU-25) was initially designed. Then, fluorophore molecule tris(2,2′-bipyridine) dichlororuthenium(II) hexahydrate (Ru(bpy)32+) was loaded onto HPU-25 by a simple post-synthesis strategy to prepare a new ratio fluorescence sensor Ru(II)@HPU-25. As a sensor, it can detect 2,6-pyridinedicarboxylic acid (DPA) content in real-time, with high sensitivity and selectivity, without being disturbed by probe concentration and environment. Evident changes in fluorescence color and intensity are observed in distinguishing DPA contents. In addition, after detecting DPA, it can be used for consecutive visual detection of dichromate anion (Cr2O72−) in water. This multifunctional fluorescence sensor could make up for the defects of single fluorescence and single function of the traditional fluorescence sensor by using the dual strategies of ratio sensing and cascade sensing. Interestingly, DPA and Cr2O72− in samples can be visually detected rapidly in real-time through the chroma acquisition APP on smartphone. More importantly, we also designed the combination of two logic gate operations for sensing the two targets. In the poison detection in daily life, these intelligent applications of fluorescence sensors have a broad development space.

Differential sensitization toward lanthanide metal–organic framework for detection of an anthrax biomarker

A novel Tb-doped Eu-based metal-organic framework (Eu-MOF@Tb) has been developed by incorporating hexanuclear europium cluster and 2,2'-bipyridine-5,5'-dicarboxylic acid as well as coordination with Tb(III). Owing to the diverse coordination status of Tb(III) and Eu(III) in MOF, antenna effect emission from Tb(III) can be invoked by dipicolinic acid (DPA), but the luminescence originating from Eu(III) remains unchanged. Taking advantage of this phenomenon, a ratiometric luminescent method for detection of DPA, a biomarker for Bacillus subtilis spores, was developed through differential sensitization toward lanthanide ions. This analysis method allowed for the detection of DPA in the 0.2-10 μM concentration range, with a detection limit of 60nM. It was further validated by spiked recoveries (89.3-110%) of real-world samples with RSD values in the range 3.9-11%. Alongside this, a paper indicator test was prepared for naked-eye detection of DPA via a dose-sensitive color evolution from red to green under UV light. The effectiveness of the proposed approach was explored in the detection of bacterial spores in real biological and environmental samples and indicated great potential for applications as a real-time monitoring system against the anthrax threat.

High sensitivity enhancement of multi-shaped silver-nanoparticle-decorated hydrophilic PVDF-based SERS substrates using solvating pretreatment

We report an extremely sensitive and ultra-low-cost PVDF-based SERS substrate using an easy fabrication process that can be conducted in a laboratory and scaled up for industrial applications. Hydrophilic PVDF membranes were sequentially dipped in ethanol and aqueous sodium chloride solution, following which multi-shaped Ag nanoparticles were deposited on the surface of the membranes by suction filtration. The simple solvating pretreatment led to an approximately 188-fold enhancement in the SERS signal intensity of R6G. Using the hydrophilic PVDF-based SERS substrate, subnanomolar sensitivity for R6G was achieved using 3 μL of sample volume on a 3×3 mm2 SERS substrate within 1 min detection time and without the need for concentration or drying processes. To demonstrate the feasibility of the developed SERS substrate as a sensor for bacterial spore detection, it was applied for the detection of the representative biomarker dipicolinic acid (DPA). The limits of detection of DPA and Bacillus spores were measured as 1 ppm and 5×103 spores/mL, respectively. The optimized configuration of low substrate area and wet micro-analyte volume makes the PVDF-based SERS substrate a simple and low-cost tool for the trace-level detection of small molecules in a rapid, sensitive, and high-throughput manner.

A Review of Piezoelectric and Magnetostrictive Biosensor Materials for Detection of COVID-19 and Other Viruses.

The spread of the severe acute respiratory syndrome coronavirus has changed the lives of people around the world with a huge impact on economies and societies. The development of wearable sensors that can continuously monitor the environment for viruses may become an important research area. Here, the state of the art of research on biosensor materials for virus detection is reviewed. A general description of the principles for virus detection is included, along with a critique of the experimental work dedicated to various virus sensors, and a summary of their detection limitations. The piezoelectric sensors used for the detection of human papilloma, vaccinia, dengue, Ebola, influenza A, human immunodeficiency, and hepatitis B viruses are examined in the first section; then the second part deals with magnetostrictive sensors for the detection of bacterial spores, proteins, and classical swine fever. In addition, progress related to early detection of COVID‐19 (coronavirus disease 2019) is discussed in the final section, where remaining challenges in the field are also identified. It is believed that this review will guide material researchers in their future work of developing smart biosensors, which can further improve detection sensitivity in monitoring currently known and future virus threats.

Dual-emission of silicon nanoparticles encapsulated lanthanide-based metal-organic frameworks for ratiometric fluorescence detection of bacterial spores.

Dipicolinic acid (DPA) is employed as a significant biomarker to detect Bacillus anthracis, which can do serious damages to the health of human beings. Hence, it is crucial to develop a fast and highly efficient strategy for DPA monitoring. In this work, based on silicon nanoparticles (Si NPs) and terbium metal-organic frameworks (Tb-MOFs), a hybrid structure (Si NPs/Tb-MOFs) as a novel dual-emitting fluorescence probe was fabricated for ratiometric detection of DPA, where blue light-emitting Si NPs (Ex: 280nm; Em: 422nm) are encapsulated into green light-emitting Tb-MOFs (Ex: 280nm; Em: 547nm). The optical properties and chemical composition of the as-obtained Si NPs/Tb-MOFs were characterized in detail. The Si NPs/Tb-MOFs probe not merely possesses the merits of a facile synthesis method but also is an excellent fluorescence probe. The response time towards DPA is less than 30s, revealing that the process of detecting DPA can be completed in such a short time. The limit of detection for DPA is 5.3nM, which is four orders of magnitude lower than an infectious dosage of anthrax spores for human beings (60μM). This dual-emitting Si NPs/Tb-MOFs probe with interference-free and self-calibrating properties may be a potential candidate for further development in medical diagnosis. Graphical abstract.

Fluorescence of copper(I) and mixed valence copper(I/II) complexes with dipicolinic acid and their catalytic activity on catechol oxidation

The pyridine-2,6-dicarboxylic acid or dipicolinic acid (H2DPA) is a unique biomarker for the detection of bacterial spores. Therefore the qualitative and quantitative determination of H2DPA is crucial for the timely detection of bacteria. Here, the first analysis of H2DPA which is based on the fluorescence of coinage metal complexes is reported. Catechol oxidase, on the other hand, is a copper containing oxidase enzyme that catalyzes the oxidation of ortho-diphenols into ortho-quinones. The catechol oxidation reaction is used in the determination of catecholamines, adrenaline, noradrenaline, dopamine, etc. Therefore, the development of new copper complexes which act as catechol oxidase models can be applied in the detection of these hormonally (adrenaline, noradrenaline, dopamine, etc.) active compounds. Thus, the copper(I) complex of dipicolinic acid (H2DPA) with formulae [CuI(ΗDPA)(ΤPP)2] ·H2O (1) (TPP = triphenylphosphine) and the mixed valence copper(I/II) one with formulae {[CuII(DPA)(H2DPA)] · [CuI(TPP)3]2}·2DMSO (2) have been synthesized and characterized by m.p., FT-IR, UV–vis, Fluorescence and 1H NMR spectroscopic techniques and X-ray crystallography. These complexes (1–2) and the known ones [CuΙI(DPA)(H2DPA)] (3) and [Cu2Cl2(TPP)3] (4) were used for the evaluation of their fluorescence properties. The excitation wavelengths maxima are observed at 350 nm while they emit at 407–496 nm. The catalytic activities of 1–3 on the oxidation of 3,5-di-tertbutyl-o-catechol to 3,5-di-tert-butyl-o-quinone were also studied spectrophotometrically. The high yields of 2 and 3 suggest their biomimetic behavior with catechol oxidase.

Model of microwave effects on bacterial spores

A recent application of microwaves in the healthcare area is for the rapid detection of bacterial spores, particularly of clinically significant spores such as Clostridium difficile. Here, we present a working model of C. difficile spore disruption by the action of a 2.45 GHz microwave electric field, independent of overall sample heating. The model shows how inner layers of the spore with the lower complex permittivity values will be subject to higher values of the electric field. The model also shows how the electric effects can be enhanced, focusing the electric field into “hotspots” using “angled” nanoparticles, yielding effective DNA release even at low microwave power levels. The model’s predictions have been validated through experimental results, which show an enhancement of DNA release from spores.

Perturbing Tandem Energy Transfer in Luminescent Heterobinuclear Lanthanide Coordination Polymer Nanoparticles Enables Real-Time Monitoring of Release of the Anthrax Biomarker from Bacterial Spores.

Lanthanide-based luminescent sensors have been widely used for the detection of the anthrax biomarker dipicolinic acid (DPA). However, mainly based on DPA sensitization to the lanthanide core, most of them failed to realize robust detection of DPA in bacterial spores. We proposed a new strategy for reliable detection of DPA by perturbing a tandem energy transfer in heterobinuclear lanthanide coordination polymer nanoparticles simply constructed by two kinds of lanthanide ions, Tb3+ and Eu3+, and guanosine 5'-monophosphate. This smart luminescent probe was demonstrated to exhibit highly sensitive and selective visual luminescence color change upon exposure to DPA, enabling accurate detection of DPA in complex biosystems such as bacterial spores. DPA release from bacterial spores on physiological germination was also successfully monitored in real time by confocal imaging. This probe is thus expected to be a powerful tool for efficient detection of bacterial spores in responding to anthrax threats.

Terbium Functionalized Micelle Nanoprobe for Ratiometric Fluorescence Detection of Anthrax Spore Biomarker.

Rapid, sensitive, and selective quantitative detection of pyridine dicarboxylic acid (DPA) as biomarker of anthrax spores is in great demand since anthrax spores are highly lethal to human beings and animals and also potential biological warfare agents. Herein, we prepared a ratiometric fluorescence lanthanide functionalized micelle nanoprobe by "one-pot" self-assembly, with an amphiphilic ligand containing β-diketone derivative which can "immobilize" terbium ions through the coordination interaction and a fluorophore as fluorescence reference (FR). The detection strategy was ascribed to Tb3+ ions in lanthanide functionalized micelle, which can be sensitized to emit the intrinsic luminescence upon addition of DPA due to the presence of energy transfer when DPA chromophore coordinated with Tb3+ ion. The fluorescence intensity of FR remained essentially constant, leading to ratiometric fluorescence response toward DPA. The results demonstrate that the terbium functionalized micelle was able to sensitively detect DPA with a linear relation in the range of 0 μM to 7.0 μM in aqueous solution, which also showed remarkable selectivity to DPA over other aromatic ligands. Our work paves a new way in the design of ratiometric fluorescence lanthanide functionalized micelle nanoprobes which can be promising for selective and sensitive detection of bacterial spores or biomolecules.

Facile ratiometric fluorapatite nanoprobes for rapid and sensitive bacterial spore biomarker detection

Since bacterial spores, such as Bacillus anthracis spores, are extremely hazardous to human beings and animals, efforts have focused on the development of bacterial spore detector with rapid response and high selectivity and sensitivity. Therefore, we reported a facile one-step chelating-reagent-assisted hydrothermal synthesis of lanthanide-doped fluorapatite (FA) nanoprobes for detecting the biomarker of bacterial spores. In FA synthesis, ethylenediaminetetraacetic acid (EDTA) can serve not only as a shape controller and a stabilizer but also as a chelating reagent for lanthanide ions. After the fabrication, terbium (or europium) ions were not only embedded into lattice models of FA nanocrystals, but also chelated by EDTA on the surface of the FA nanocrystals, which can be available for detecting the biomarker (i.e., dipicolinic acid) of bacterial spores. The obtained FA: Ln–EDTA nanoprobes can provide reference fluorescence which play crucial roles in the calibration and correction of concentration. The fluorescence intensities of FA: Tb–EDTA or FA: Eu–EDTA from Tb3+ or Eu3+ emission were highly sensitive and increased gradually with increasing DPA concentrations. The limit of detection of FA: Tb–EDTA was 8.2nM, which is lower than that of FA: Eu–EDTA (20.9nM). It can be attributed to the fact that DPA can achieve a more effective energy transfer to Tb3+ than to Eu3+. Additionally, the nanoprobes were successfully applied for the determination of DPA in fetal calf serum samples. Given their rapid response and high sensitivity and selectivity, these ratiometric fluorescent nanoprobes are promising tools for the detection of bacterial spores.

Rapid onsite detection of bacterial spores of biothreat importance by paper-based colorimetric method using erbium-pyrocatechol violet complex.

Dipicolinic acid (DPA) is an important chemical marker for the detection of bacterial spores. In this study, complexes of lanthanide series elements such as erbium, europium, neodymium, and terbium were prepared with pyrocatechol violet and effectively immobilized the pyrocatechol violet (PV)-metal complex on a filter paper using polyvinyl alcohol. These filter paper strips were employed for the onsite detection of bacterial spores. The test filter papers were evaluated quantitatively with different concentrations of DPA and spores of various bacteria. Among the four lanthanide ions, erbium displayed better sensitivity than the other ions. The limit of detection of this test for DPA was 60 μM and 5 × 10(6) spores. The effect of other non-spore-forming bacteria and interfering chemicals on the test strips was also evaluated. The non-spore-forming bacteria did not have considerable effect on the test strip whereas chemicals such as EDTA had significant effects on the test results. The present test is rapid and robust, capable of providing timely results for better judgement to save resources on unnecessary decontamination procedures during false alarms.

Luminescent lanthanide graphene for detection of bacterial spores and cysteine.

Here, we describe a new approach for preparation of luminescent lanthanide graphene in the presence of dipicolinic acid (DPA). Hg(2+) can competitively bind with DPA which greatly quenches the fluorescence and the resultant complex is able to selectively and sensitively detect cysteine with a detection limit of 5 nM.

Quantitative and sensitive RNA based detection of Bacillus spores

The fast and reliable detection of bacterial spores is of great importance and still remains a challenge. Here we describe a direct RNA-based diagnostic method for the specific detection of viable bacterial spores which does not depends on an enzymatic amplification step and therefore is directly appropriate for quantification. The procedure includes the following steps: (i) heat activation of spores, (ii) germination and enrichment cultivation, (iii) cell lysis, and (iv) analysis of 16S rRNA in crude cell lysates using a sandwich hybridization assay. The sensitivity of the method is dependent on the cultivation time and the detection limit; it is possible to detect 10 spores per ml when the RNA analysis is performed after 6 h of enrichment cultivation. At spore concentrations above 106 spores per ml the cultivation time can be shortened to 30 min. Total analysis times are in the range of 2–8 h depending on the spore concentration in samples. The developed procedure is optimized at the example of Bacillus subtilis spores but should be applicable to other organisms. The new method can easily be modified for other target RNAs and is suitable for specific detection of spores from known groups of organisms.

Rapid Homogenous Time-Resolved Fluorescence (HTRF) Immunoassay for Anthrax Detection

Infection with Bacillus anthracsis spores induces an acute anthrax disease that can cause casualties and death in untreated cases. Thus rapid diagnosis of anthrax at early stage of the disease is essential to allow an effective treatment. Here we present the development of rapid and sensitive homogenous time-resolved fluorescence (HTRF) immunoassays based on the energy transfer process of europium cryptate (EuK) donor to AlexaFluor647 acceptor. The energy transfer process is limited to d < 10nm, making the HTRF an ideal assay for examination of homogenous and complex samples, since only mutual binding of the donor and acceptor antibodies to the analyte would result in positive signal. HTRF assay was developed for the detection of the bacterial Protective Antigen (PA) toxin, a serological marker that correlates with bacteremia in infected hosts, using two monoclonal anti-PA antibodies that specifically recognize two different epitopes on the PA molecule. The assay was sensitive enabling detection of 2ng/ml PA in the serum of B. anthracsis-infected rabbits in only 15min assay. Additionally, HTRF assay was developed for the detection of bacterial spores using polyclonal anti-spore antibodies that recognize many epitopes on the bacterial surface. The assay enabled the detection of 2 × 10(6) spores/ml in 30min assay and was specific, showing no cross reactivity with closely related non-virulent bacillus cereus strain. This study describes the use of the HTRF assay for the detection of both singled-epitope (proteins) and multi-epitope (particles) as rapid, simple and sensitive method that can be used at the time that fast results are needed to allow an effective medical care.

Ratiometric Luminescent Detection of Bacterial Spores with Terbium Chelated Semiconducting Polymer Dots

We report a ratiometric fluorescent sensor based on semiconducting polymer dots chelated with terbium ions to detect bacterial spores in aqueous solution. Fluorescent polyfluorene (PFO) dots serve as a scaffold to coordinate with lanthanide ions that can be sensitized by calcium dipicolinate (CaDPA), an important biomarker of bacterial spores. The absorption band of PFO dots extends to deep UV region, allowing both the reference and the sensitizer can be excited with a single wavelength (~275 nm). The fluorescence of PFO remains constant as a reference, while the Tb(3+) ions exhibit enhanced luminescence upon binding with DPA. The sharp fluorescence peaks of β-phase PFO dots and the narrow-band emissions of Tb(3+) ions enable ratiometric and sensitive CaDPA detection with a linear response over nanomolar concentration and a detection limit of ~0.2 nM. The Pdots based sensor also show excellent selectivity to CaDPA over other aromatic ligands. Our results indicate that the Tb(3+) chelated Pdots sensor is promising for sensitive and rapid detection of bacterial spores.

Nanoparticle-based substrates for surface-enhanced Raman scattering detection of bacterial spores

The development of ultrasensitive and rapid methods for the detection of bacterial spores is important for medical diagnostics of infectious diseases. While Surface-Enhanced Raman Spectroscopic (SERS) techniques have been increasingly demonstrated for achieving this goal, a key challenge is the development of sensitive and stable SERS substrates or probes. This Minireview highlights recent progress in exploring metal nanoparticle-based substrates, especially gold nanoparticle-based substrates, for the detection of biomarkers released from bacterial spores. One recent example involves assemblies of gold nanoparticles on a gold substrate for the highly sensitive detection of dipicolinic acid (DPA), a biomarker for bacterial spores such as Bacillus anthracis. This type of substrate exploits a strong SERS effect produced by the particle-particle and particle-substrate plasmonic coupling. It is capable of accurate speciation of the biomarker but also selective detection under various reactive or non-reactive conditions. In the case of detecting Bacillus subtilis spores, the limit of detection is quite comparable (0.1 ppb for DPA, and 1.5 × 10(9) spores per L (or 2.5 × 10(-14) M)) with those obtained using silver nanoparticle-based substrates. Implications of the recent findings for improving the gold nanoparticle-based SERS substrates with ultrahigh sensitivity for the detection of bacterial spores are also discussed.

Simple leaky-waveguide devices for the detection of bacterial spores

A variety of biocompatible optical leaky waveguide sensors were developed for bacterial detection. Divalent sensitive polymer waveguides incorporating the chelating agent nitrilotriacetic acid (NTA) were used to monitor the release of Ca 2+ ions from Bacillus subtilis spores during the first stage of germination. Co-polymer waveguides incorporating hydrolyzed starch were used to detect the presence of exo-enzymes released during the later stages of vegetative growth. The limit of detection for calcium, based on changes in the intensity of the TE 0 mode was 60 μM. Changes in refractive index were demonstrated by measuring the change in the angular position of the TE 1 mode resulting from the degradation of the waveguide by α-amylase.