WO2021045682A1

WO2021045682A1 - Colorimetric method for bacteria detection

Info

Publication number: WO2021045682A1
Application number: PCT/SG2020/050508
Authority: WO
Inventors: Yen Nee Tan; Yong Yu; Xinting ZHENG; Choon Peng TENG; Takuya Sato
Original assignee: Agency For Science, Technology And Research
Priority date: 2019-09-04
Filing date: 2020-09-01
Publication date: 2021-03-11

Abstract

The present invention relates to a colorimetric method for detecting bacterial cells in a sample. The colorimetric method comprises: (a) contacting the sample with nanoparticles to form a mixture, wherein the nanoparticles are characterized by an amorphous core formed from threonine and polyethylenimine; and subsequently (b) contacting the formed mixture with gold nanoparticles, wherein the gold nanoparticles provide a colorimetric indication of the bacterial cells in the sample, depending on the extent of gold nanoparticles aggregation induced by the nanoparticles of (a). In one embodiment, said nanoparticles of (a) are further characterized by a surface functionalised with at least a carbohydrate, such as mannose.

Description

COLORIMETRIC METHOD FOR BACTERIA DETECTION

Technical Field

The present invention relates, in general terms, to a colorimetric assay for detecting bacterial cells in a sample.

Background

The presence of pathogenic bacteria in food and drink poses a threat to both public health and security. According to a report by World Health Organization, about 600 million people fall ill after consuming contaminated food and 420 000 die every year, among which the pathogenic bacteria has been estimated to account for 12 out of 31 hazards leading to foodborne diseases. Salmonella, Campylobacter, and Enterohaemorrhagic Escherichia coli are among the most common bacteria pathogens that affect millions of people annually, causing severe symptoms including fever, headache, nausea, vomiting, abdominal pain and diarrhoea, and even fatal consequences. Therefore, developing a rapid assay technique for foodborne pathogens is a pressing need to combat foodborne diseases and ensure food/water security.

An ideal bacteria detection method should be fast, highly sensitive, cost-effective and simple to implement. Plate counting has been routinely adopted as a golden-standard due to its accuracy and reliability. However, it takes long time (e.g., up to 3 days) to get the results and requires well-trained personnel for cell culture. Other methods such as polymerase chain reaction (PCR) needs up to 1 day and requires specialist equipment and reagents. Much sample preparation is also required. Enzyme-linked immunosorbent assay (ELISA) need at least 3 hours but require specialized instrumentation, trained staff and complicated operation principle. Current conventional methods for bacteria detection are shown in Figure 1. The limit of detection (LOD) for plating is 1 CFU/mL, PCR is 10 CFU/mL and ELISA is 100,000 CFU/mL.

It would be desirable to overcome or ameliorate at least one of the above-described problems, or at least to provide a useful alternative.

Summary A rapid detection method for pathogenic bacteria at the lowest possible concentrations is very critical to assure food safety. Conventional bacteria detection methods, such as the plating techniques and biochemical methods, are reliable, highly robust and accurate. However, they are not particularly suitable to be used as user-friendly approaches since they require tedious culture processes, trained operators, relatively sophisticated laboratory equipment and time-consuming labours (e.g. up to 3 days). Herein is disclosed a simple, fast and ultrasensitive colorimetric method for bacteria detection in solution as illustrated in Figure 2, using biodots (or nanoparticles or nanodots) as disclosed herein to induce colorimetric changes of gold nanoparticles (AuNPs). The assay is based on the measurement of the excess free biodots in the sample solution that can cause the colour change of AuNPs to determine the bacteria concentration. In the absence of bacteria, AuNPs changes colour from red to blue due to biodots induced aggregation. While in the presence of bacteria, biodots will interact with the bacteria cells due to their unique interactions (surface binding and uptake), preventing the aggregation of AuNPs (i.e. no colour change). A low detection limit of about 1 CFU/mL of bacteria can be achieved in less than 20 mins without the need of sophisticated equipment and well-trained operators. Despite of its ultrasensitivity, the assay results can be readily observed by our naked eye, showing great potential to be used as a rapid and user-friendly solution for on-site bacteria detection.

The present invention relates to a colorimetric assay for detecting bacterial cells in a sample, including the steps of: a) contacting the sample with nanoparticles to form a first mixture; and b) subsequently contacting the first mixture with gold nanoparticles, the gold nanoparticles for providing a colorimetric indication; wherein the nanoparticles (from step (a)) are characterised by an amorphous core, the core formed from threonine and polyethylenimine (PEI); wherein a red coloration after step (b) is indicative of the presence of bacterial cells in the sample; and wherein a blue or purple coloration after step (b) is indicative of the absence of bacterial cells in the sample.

In some embodiments, the nanoparticle is further characterised by a surface, the surface functionalised with at least a carbohydrate.

In some embodiments, the carbohydrate is mannose. In some embodiments, the PEI in the nanoparticle core is a branched PEI.

In some embodiments, the amorphous core of the nanoparticle has a molecular weight of less than 3 kDa.

In some embodiments, the nanoparticle has a mean diameter of about 1 nm to about 8 nm. In some embodiments, the nanoparticle has a zeta potential of more than about +5 mV.

In some embodiments, the nanoparticle has a minimum inhibitory concentration (MIC) value against bacterial cells of more than 30 pg/mL. In some embodiments, step (a) is performed for a period of at least 10 min.

In some embodiments, step (a) is performed at a temperature of about 15 °C to about 35 °C. In some embodiments, the nanoparticle is provided at a concentration of at least 0.15 pg/mL.

In some embodiments, the gold nanoparticle has a mean particle size of about 10 nm to about 20 nm.

In some embodiments, the gold nanoparticle is passivated with citrate ions.

In some embodiments, the colorimetric indication is obtainable within at least 15 min. In some embodiments, the method further includes a step (c) of quantifying the bacterial cells in the sample after step (b).

In some embodiments, step (c) comprises determining an absorbance ratio of the gold nanoparticle at wavelengths of 630 nm and 525 nm, and comparing the absorbance ratio with a calibration plot. In some embodiments, the assay has a detection limit of at least 1 CFU/mL of bacterial cells.

Brief description of the drawings

Embodiments of the present invention will now be described, by way of non-limiting example, with reference to the drawings in which:

Figure 1 illustrates conventional methods for bacteria detection and their limitations; Figure 2 illustrates assay design for colorimetric detection of bacteria based on unique interactions among bacterial cells, antimicrobial agent (e.g., antibiotics)/biodots and gold nanoparticles (AuNPs). In the presence of lesser bacteria, the excess biodots will induce aggregation of AuNPs, leading to distinct color change from red to blue, resulting in ultrasensitive detection of bacteria cells down to 1 CFU/mL;

Figure 3 is a schematic illustration of the design of colorimetric bacteria assay based on biodots-induced aggregation of gold nanoparticles and their interactions with different amount of bacteria;

Figure 4 illustrates colorimetric assay results of E. coli with SPdots with varied duration of SPdots interacting with bacteria (A, B, C: 15, 30, and 45 min, respectively). D shows the correlation of AuNPs aggregation with E coli concentration at SPdots concentration of 3.58 pg/mL and with 30 min incubation time of SPdots and E coli prior to addition of AuNPs. Results were obtained by taking average of duplicates and calculating the standard deviation;

Figure 5 illustrates colorimetric assay results of E coli with TPdots with varied duration of TPdots interacting with bacteria (A, B, C: 15, 30, and 45 min, respectively). D shows the correlation of AuNPs aggregation with E coli concentration at TPdots concentration of 0.179 pg/ml and with 45 min incubation time of TPdots and E coli prior to addition of AuNPs;

Figure 6 illustrates colorimetric assay results of E. coli with TPMdots with varied duration of TPMdots interacting with bacteria (A, B, C: 15, 30, and 45 min, respectively). D shows the correlation of AuNPs aggregation with E. coli concentration at TPMdots concentration of 0.179 pg/mL and with 15 min incubation time of TPMdots and E coli prior to addition of AuNPs; and

Figure 7A-E shows colorimetric detection of E. coli down to 1 CFU/mL of bacteria cells. (A) Image of mixtures of AuNPs, TPMdots and E. coli. (B to E) Relative aggregation state of AuNPs in the presence of E. coli of different concentrations at specific fixed TPMdots concentrations.

Detailed description

In this disclosure, a fast and ultrasensitive colorimetric method for bacteria detection in solution was envisioned. This is without the need of special instrument such as PCR machine and/or additional light source to excite the fluorophores for signal measurement in the conventional ELISA assay. The assay principle is illustrated in Figure 2 using biodots (nanoparticles) and gold nanoparticles as the nanoreagents for bacteria detection. The inventors have found that the biodots as disclosed herein are particularly advantageous as they can interact with bacteria cells (surface binding and uptake) and also aggregate gold nanoparticles (AuNPs) to cause the colour of solution change from red to blue. By using the biodots as the basis of an assay, the measurement of the excess/free biodots in the sample solution that can cause the colour change of AuNPs to determine the bacteria concentration. In the absence of bacteria, AuNPs changes colour due to biodots induced aggregation. While in the presence of bacteria, the aggregation of AuNPs induced by biodots is suppressed due to the unique interaction between bacteria and biodots (surface binding and cellular uptake) and thus the characteristic red colour of AuNPs solution is retained. A low detection limit of 1 CFU/mL of bacteria can be achieved in less than 20 minutes without the need of sophisticated equipment and well-trained operators. In addition to its ultrasensitivity, the assay results can be readily observed by naked eye, showing great potential to be used as a rapid and user-friendly solution for on-site bacteria detection.

Accordingly, the present invention is predicated on bacteria-biodots interactions to eliminate excessive biodots that induce undesirable AuNPs aggregation. In this regard, the biodots are incubated with analyte (bacteria) first and followed by addition of AuNPs.

Without wanting to be bound by theory, the inventors believe that a colorimetric change can be based on the favourable uptake of the biodots into the bacterial cells. To this end, it was found in contrast to other crystalline nanoparticles which tend to reside on the surface of bacterial cells and are toxic to cells, the amorphous nanoparticles (or biodots) as disclosed herein are advantageously taken up into the bacterial cells due to their small size, high affinity ligand present on the biodot surface and low toxicity. The endocytosis of these biodots are also relatively quick, such that an assay based on this interaction can yield results in a short amount of time.

The inventors have found that the aggregation of colorimetric nanoparticles such as gold nanoparticles can be used advantageously as an indication of the presence of bacterial cells. Toward this end, rather than suppressing the aggregation of AuNPs, the natural tendency of AuNPs to aggregate can be used to indicate the presence or absence of bacterial cells.

Even when the biodots are surface bound to the bacterial cells and are partially exposed, it was found that when AuNPs are in close proximity, no aggregation of AuNPs was observed, thus allowing for a high accuracy of the assay.

In contrast, other bacterial cell assays which uses only one type of nanoparticles have a higher detection limit and lower accuracy compared to the presently disclosed assay. This is believed to be due to the direct interaction of the nanoparticles with the bacterial cells, which is an uncontrollable factor. As the nanoparticles are also subsequently used in an analysis step, the error from the previous cell interaction step is carried over in the analysis. By separating the cell interaction step and the analysis step (as shown in the present invention), a lower detection limit and higher accuracy can be obtained. In addition, unlike prevailing nanoparticle sensor systems in which the amount of analyte is directly correlated to the extent of aggregation, the amount of bacteria in the current technology is inversely correlated to the extent of aggregation, i.e., less bacteria corresponds to more severe aggregation, which is more favourable for detecting analytes with a very low concentration. This allows for a better limit of detection (LOD) and higher accuracy.

The present invention relates to a colorimetric assay for detecting bacterial cells in a sample, including the steps of: a) contacting the sample with nanoparticles to form a first mixture; and b) subsequently contacting the first mixture with gold nanoparticles, the gold nanoparticles for providing a colorimetric indication; wherein the nanoparticles (from step (a)) are characterised by an amorphous core, the core formed from an amino acid and polyethylenimine (PEI); wherein a red coloration after step (b) is indicative of the presence of bacterial cells in the sample; and wherein a blue or purple coloration after step (b) is indicative of the absence of bacterial cells in the sample.

In some embodiments, the core of the nanoparticles (or biodot) is formed from an amino acid, the amino acid is selected from serine and threonine. In other embodiments, the amino acid is threonine.

Accordingly, in some embodiments, the colorimetric assay for detecting bacterial cells in a sample includes the steps of: a) contacting the sample with nanoparticles to form a first mixture; and b) subsequently contacting the first mixture with gold nanoparticles, the gold nanoparticles for providing a colorimetric indication; wherein the nanoparticles (from step (a)) are characterised by an amorphous core, the core formed from threonine and polyethylenimine (PEI); wherein a red coloration after step (b) is indicative of the presence of bacterial cells in the sample; and wherein a blue or purple coloration after step (b) is indicative of the absence of bacterial cells in the sample.

As used herein, nanoparticles or nanodots or biodots refer to particles of matter having any shape with dimensions in the range of about 1 x 10^-9 m and about 1 x 10^-7 m. The average particle size can be equivalent to the mean diameter of the nanoparticle. The present definition also includes anisotropic nanoparticles. Such anisotropic nanoparticles can, for example, include non-spherical nanoparticles, nanorods, nanocubes, nanochains, nanostars, nanoflowers, nanoreefs, nanowhiskers, nanofibers, and nanoboxes.

Amorphous refers to a non-crystalline solid that lacks the long-range order that is characteristic of a crystal. Amorphous materials have an internal structure made of interconnected structural blocks. These blocks can be similar to the basic structural units found in the corresponding crystalline phase of the same compound. Whether a material is liquid or solid depends primarily on the connectivity between its elementary building blocks so that solids are characterized by a high degree of connectivity whereas structural blocks in fluids have lower connectivity. Amorphous nanoparticles can have some short range order at the atomic length scale due to the nature of chemical bonding. Furthermore, in very small nanoparticles relaxation of the surface and interfacial effects distort the atomic positions, decreasing the structural order. The skilled person would understand that to characterise amorphous material (as compared to non-amorphous or crystalline material), various techniques such as X-ray absorption spectroscopy (XAS), X-ray diffraction (XRD), and inductively coupled plasma (ICP) can be used. For example, long-range structural order peaks from XRD are expected to be minimal or non-definable in amorphous material.

In some embodiments, the core is formed from threonine and polyethylenimine (PEI). In other embodiments, threonine and polyethylenimine (PEI) are covalently bonded to each other via amide linkages. In some embodiments, the PEI is a branched PEI. Accordingly, a matrix structure is formed when the branched PEI cross-linked with threonine.

It was found that a branched PEI is particularly advantageous for facilitating formation of covalent bonds with amino acids such as threonine and thus form a more compact nanoparticle core. In this way, the density of crosslinking is increased.

In some embodiments, the PEI or branched PEI has a molecular weight of about 600. In other embodiments, the molecular weight is about 1,000, about 2,500, about 5,000, about 10,000, about 25,000, about 50,000, about 75,000, about 100,000, about 200,000 or about 300,000. The molecular weight can be determined using analytical methods such as liquid chromatography and/or gel permeation chromatography.

The PEI can have a structure as shown in Formula (II):

The branched PEI can have a structure as shown in Formula (III):

Depending on the molecular weight, the integer n can be determined accordingly.

The PEI and branched PEI (and its salts thereof) as described herein also includes functionalised PEI and functionalised branched PEI. For example, the PEI and branched PEI can be modified with stearic acid, polyethylene glycol (PEG), and hydroxyl group. In this way, the nanoparticle can have other functionalities as imparted by the PEI.

In some embodiments, the nanoparticle is characterised by an amorphous core having a surface.

In some embodiments, the core has a molecular weight of less than 3 kDa. In other embodiments, the molecular weight is less than 2.5 kDa, less than 2 kDa, less than 1.5 kDa or less than 1 kDa. The molecular weight of the core can be tuned by varying the synthetic conditions. For example, the hydrothermal reaction can be tuned by varying the temperature, pressure and the reaction time.

To this end, the inventors have found that a nanoparticle with a core formed from threonine and PEI is particularly advantageous. Without wanting to be bound by theory, it is believed that the core formed from a hydrothermal reaction can trigger the precursors (i.e. threonine and PEI) to undergo polymerization or crosslinking. It is believed that the combination of threonine and PEI allows for a further rearrangement and finally aromatize into conjugated pi system. Such nanoparticle are able to bind to bacteria more efficiently and subsequently being taken up by the cells, so more threonine-PEI dots can bind to the same amount of bacteria thus giving higher accuracy to the assay.

Further, threonine-PEI dots in contrast to serine-PEI dots exhibit different size distributions. Particularly advantageously, the size distribution of threonine-PEI dots is such that the biodots can interact with gold nanoparticles and bacteria more efficiently to give an assay with a larger working range at a lower concentration.

In some embodiments, the surface of the nanoparticle is functionalised with at least a carbohydrate.

Functionalisation with a carbohydrate is particularly advantageous as specific bacterial cells can be targeted when used in an assay. Compared to unfunctionalised nanoparticles, carbohydrate functionalised nanoparticles are able to be endocytosised to a larger extend without causing the bacterial cells to undergo apoptosis. Additionally, unspecific binding is largely reduced. This allows for the bacteria assay to be more accurate and/or precise.

As used herein, carbohydrate refers to a biomolecule consisting of carbon (C), hydrogen (H) and oxygen (O) atoms. Carbohydrates can have a hydrogen -oxygen atom ratio of 2:1 and thus have an empirical formula Cm(H20)n (where m may be different from n). This formula holds true for monosaccharides. Some exceptions exist; for example, deoxyribose has the empirical formula C5H 10O4; these are also included within the scope of carbohydrate. The saccharides are divided into four chemical groups: monosaccharides, disaccharides, oligosaccharides, and polysaccharides. Examples of carbohydrates include, but is not limited to, fructose, glucose, galactose, xylose, sucrose, lactose, maltose, trehalose, sorbitol, mannitol, maltodextrins, raffinoise, stachyose, fructo-oligosaccharides, amylose, amylopectin, modified starches, glycogen, cellulose, hemicellulose, pectins and hydrocolloids. Stereoisomers such as diastereomers or epimers of carbohydrates are also included within this scope.

In some embodiments, the carbohydrate is mannose. As shown herein, functionalisation with mannose is particularly advantageous for sensing of E. coli. Depending on the bacteria type, other carbohydrates can be used. For example, the carbohydrate can be selected from fructose, glucose, galactose, xylose, sucrose, lactose, maltose, trehalose, sorbitol, and mannitol.

In some embodiments, the bacterial cell assay is for detecting E.coli. In this embodiment, the nanoparticles have a surface functionalised with mannose. Advantageously, the lipopolysaccharide of E. coli (gram-negative bacteria) was found to bind with mannose specifically, thus mannose can enhance the surface interaction/uptaking of the biodots by bacterial cells and further improve the assay performance.

Mannose is a sugar monomer of the aldohexose series of carbohydrates. It is a C-2 epimer of glucose. Mannose commonly exists as two different-sized rings, the pyranose (six-membered) form and the furanose (five-membered) form. Each ring closure can have either an alpha or beta configuration at the anomeric position, and rapidly undergoes isomerization among these four forms. These configurations, including the linear configurations, are included within the scope of the invention.

In some embodiments, the nanoparticle has a mean diameter of about 1 nm to about 8 nm. In other embodiments, the nanoparticle has a mean diameter of about 1 nm to about 7 nm, 1 nm to about 6 nm, 1 nm to about 5 nm, 1 nm to about 4 nm, or 1 nm to about 3 nm. The mean diameter can be tuned by varying the synthetic conditions. For example, the hydrothermal process can be tuned by varying the temperature, pressure and reaction time.

In some embodiments, the nanoparticle has a zeta potential of more than about +5 mV. In other embodiments, the zeta potential is more than about +7.5 mV, more than about + 10 mV, or more than about +15 mV. The zeta potential may be varied by, for example, varying the amount of carbohydrate on the surface of the nanoparticles. For example, by using a lesser amount of carbohydrate during the synthesis, a higher zeta potential for the nanoparticles can be obtained. This can be advantageous when a fast initial interaction with the membrane of the bacterial cell is desired as bacterial cell membrane can have a net negative charge.

In some embodiments, the nanoparticle has a minimum inhibitory concentration (MIC) value against bacteria of more than 30 pg/mL. The minimum inhibitory concentration (MIC) is the lowest concentration of a chemical which prevents visible growth of a bacterium or bacteria. MIC depends several factors, such as the microorganism and the compound (or nanoparticle). In other embodiments, the nanoparticle has a minimum inhibitory concentration (MIC) value against E. coli of more than 30 pg/mL, more than 40 pg/mL, more than 50 pg/mL, more than 60 pg/mL, more than 70 pg/mL, more than 80 pg/mL, or more than 90 pg/mL. In other embodiments, the nanoparticle has a minimum inhibitory concentration (MIC) value against S. aureus of more than 30 pg/mL, more than 60 pg/mL, or more than 90 pg/mL.

Alternatively, the nanoparticle can have a IC50 value. The half maximal inhibitory concentration (IC50) is a measure of the potency of a substance (or nanoparticle) in inhibiting a specific biological or biochemical function. IC50 is a quantitative measure that indicates how much of a particular inhibitory substance is needed to inhibit, in vitro, a given biological process or biological component by 50%. The IC50 value will correspondingly be half that of the MIC value. The nanoparticle can be maintained as a dispersion or suspension in an appropriate medium in order to form a nanoparticle reagent for use in an assay. Examples of such medium are aqueous medium. For example, water, phosphate buffered saline or other biological buffers can be used for suspending the nanoparticles. Alternatively, the nanoparticles can be freeze-dried as powder for reconstitution when use in an assay. The reconstituted nanoparticles can be prepared as a concentrated sample and further diluted to various different concentrations before adding to the bacteria sample. The term 'aqueous medium¹ used herein refers to a water based solvent or solvent system, and which comprises of mainly water. Such solvents can be either polar or nonpolar, and/or either protic or aprotic. Solvent systems refer to combinations of solvents which resulting in a final single phase. Both 'solvents' and 'solvent systems' can include, and is not limited to, pentane, cyclopentane, hexane, cyclohexane, benzene, toluene, dioxane, chloroform, diethylether, dichloromethane, tetrahydrofuran, ethyl acetate, acetone, dimethylformamide, acetonitrile, dimethyl sulfoxide, nitromethane, propylene carbonate, formic acid, butanol, isopropanol, propanol, ethanol, methanol, acetic acid, ethylene glycol, diethylene glycol or water. Water based solvent or solvent systems can also include dissolved ions, salts and molecules such as amino acids, proteins, sugars and phospholipids. Such salts may be, but not limited to, sodium chloride, potassium chloride, ammonium acetate, magnesium acetate, magnesium chloride, magnesium sulfate, potassium acetate, potassium chloride, sodium acetate, sodium citrate, zinc chloride, HEPES sodium, calcium chloride, ferric nitrate, sodium bicarbonate, potassium phosphate and sodium phosphate. As such, biological fluids, physiological solutions and culture medium also falls within this definition.

The nanoparticle reagent can additional comprise other excipients. For example, an excipient can be added to improve the shelf-life of the nanoparticles. Examples of excipients are, but not limited to, salts such as NaCI, KCI, Na2FIP04, KFhPCU and small molecules such as TAPS ([tris(hydroxymethyl)methylamino]propanesulfonic acid), Bicine (2-(bis(2-hydroxyethyl)amino)acetic acid), Tris

(tris(hydroxymethyl)a mi nomethane) or (2-amino-2-(hydroxymethyl)propane-l,3-diol), Tricine (N-[tris(hydroxymethyl)methyl]glycine), TAPSO (3-[N- tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid), FIEPES (4-(2- hydroxyethyl)-l-piperazineethanesulfonic acid), TES (2-[[l,3-dihydroxy-2- (hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid), MOPS (3-(N- morpholino)propanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), Cacodylate (dimethylarsenic acid), and MES (2-(N-morpholino)ethanesulfonic acid).

In some embodiments, the sample comprises bacterial cells selected from gram negative-bacterial cells and gram-positive bacterial cells. In other embodiments, the sample comprises bacterial cells selected from E. coli and S. aureus. In other embodiments, the sample comprises Gram-positive bacterial cells selected from S. saprophyticus, S. bovis, C. urealyticum, S. aureus, B. cereus, S. pneumonia, A. adiacens, S. mitis group, S. agalactiae, S. lugdunensis, C. jelkenium, Lactobacillus sp., C. septicum, Veillonella, Eubacterium, Clostridium sp., C. difficile, C. perfringens, Listeria monocytogenes, Erysipelothrix rhusiopathiae, Arcanobacterium bemolyticum, Bacillus megaterium, Bacillus subtilis, Brevibacterium linens, Corynebacterium glutamicum, Mycobacterium smegmatis, Rhodococcus erythropolis, and Streptomyces lividans. In other embodiments, the sample comprises Gram-negative bacterial cells selected from Proteus spp., Escherichia coli, Morganella, N. gonorrhoea, Moraxella catarrhalis, N. meningitides, Klebsiella pneumonia, Aeromonas hydrophila, Providencia, Enterobacter cloacae, Cariobacter hominis, H. influenza, Alkaligenes, Burkholderia cepacia, H. parainfluenzae, Pasturella multocida, Kingella kingae, Brucella sp., C. jejuni, Haemophilus aphrophilus, Pseudomonas, and Stenotrophomonas maltophilia, Agrobacterium tumefaciens, Azospirillum brasilense, Bordetella avium, Brevundimonas diminuta, Burkholderia cepacia, Burkholderia gladioli, Burholderia vietnamensis, Chromobacterium violaceum, Citrobacter freundii, Enterbacter aerogenes, Erwinia amylovora, Erwinia carotovora, Proteus vulgaris, Rhizobium etli, Salmonella enteritidis, Serratia entomophila, Shigella flexneri, Sphingomonas wittichii, Variovorax paradoxus, Vibrio harveyi, Xanthomonas, and Yersinia enterocolitica. In some embodiments, the bacterial cell is foodborne pathogen selected from Clostridium botulinum, Campylobacter jejuni, Escherichia coli, Listeria monocytogenes, Salmonella, Shiga toxin-producing E. coli such as verocytotoxic E. coli (VTEC) or enterohemorrhagic E. coli (EHEC), Shigella, Staphylococcus aureus, and Vibrio parahaemolyticus. The skilled person would understand that to target other bacterial cells, the carbohydrate on the surface of the amorphous nanoparticle can be varied to improve the detection limit of the assay for other bacterial cells.

In some embodiments, step (a) is performed for a period of at least 10 min. This allows for sufficient time for the nanoparticles to interact with the bacterial cells in the sample. In other embodiments, the time is at least 15 min, at least 20 min, at least 30 min, at least 40 min, at least 60 min, or at least 80 min.

In some embodiments, step (a) is performed at ambient temperature. In other embodiments, the temperature is about 15 °C to about 35 °C.

In some embodiments, the nanoparticle is provided at a concentration of at least 0.15 pg/mL. In other embodiments, the concentration is at least at least 0.16 pg/mL, at least 0.17 pg/mL, at least 0.18 pg/mL, at least 0.19 pg/mL, at least 0.2 pg/mL, 0.3 pg/mL, 0.5 pg/mL, 0.75 pg/mL, 1 pg/mL, 1.5 pg/mL, 2 pg/mL, 2.5 pg/mL, 3 pg/mL, 3.5 pg/mL, or 4 pg/mL.

In some embodiments, the nanoparticle is provided at a concentration of about 0.1 pg/mL to about 0.2 pg/mL. In other embodiments, the nanoparticle is provided at a concentration of about 0.15 pg/mL to about 0.2 pg/mL.

In some embodiments, when serine-PEI nanoparticles (SPdots) are used, the nanoparticle is provided at a concentration of about 3.5 pg/mL and is contacted with the sample for about 30 min. In other embodiments, the nanoparticle is provided at a concentration of about 2 pg/mL to about 3 pg/mL. In other embodiments, the nanoparticle is provided at a concentration of about 2.5 pg/mL to about 3 pg/mL.

In some embodiments, when threonine-PEI nanoparticles (TPdots) are used, the nanoparticle is provided at a concentration of about 0.17 pg/mL and is contacted with the sample for about 45 min. In other embodiments, the nanoparticle is provided at a concentration of about 0.15 pg/mL to about 0.2 pg/mL.

In some embodiments, when threonine-PEI-mannose nanoparticles (TPMdots) are used, the nanoparticle is provided at a concentration of about 0.17 pg/mL and is contacted with the sample for about 15 min. In other embodiments, the nanoparticle is provided at a concentration of about 0.15 pg/mL to about 0.2 pg/mL.

In some embodiments, the sample comprises about 1 x 10⁴ CFU/mL to about 1 x 10⁹ CFU/mL of bacterial cells. In other embodiments, the sample comprises about 5 x 10⁴ CFU/mL to about 1 x 10⁹ CFU/mL of bacterial cells, about 1 x 10⁵ CFU/mL to about 1 x 10⁹ CFU/mL of bacterial cells, about 1 x 10⁵ CFU/mL to about 5 x 10⁸ CFU/mL of bacterial cells, or about 1 x 10⁵ CFU/mL to about 1 x 10⁸ CFU/mL of bacterial cells.

If the sample containing bacterial cells falls outside and above the range as mentioned above, serial dilutions by a factor of, for example, 10 times can be done to lower the concentration of bacteria cells, if required. For example, if a sample that is serially diluted 4 times is detectable within the calibration range and its concentration is determined as 10^s CFU/mL by the assay, the original concentration can be calculated back as 10¹¹ CFU/mL.

If the sample containing bacterial cells falls outside and below the range as mentioned above, the bacterial cells can, for example, be concentrated from the initial solution and reconstitute to higher concentrations for detection.

The first mixture of bacterial cells and nanoparticles are subsequently contacted with a gold nanoparticle (AuNP). The gold nanoparticle provides a colorimetric indication in order to make a determination about the presence of bacterial cells in the sample. For example, the colorimetric indication allows the determination to be made by eye. A red or purple coloration indicates the presence of bacterial cells while a blue coloration indicates the absence of bacterial cells.

Advantageously, a purification step is not required, thus saving time and making the assay easy to use.

In some embodiments, the gold nanoparticle has a mean particle size of about 10 nm to about 20 nm. In other embodiments, the gold nanoparticle has a mean particle size of about 12 to about 20 nm, about 12 to about 18 nm, or about 12 to about 16 nm.

In some embodiments, the gold nanoparticle is passivated with citrate ions. In other embodiments, the gold nanoparticle is passivated with cetyl trimethyl ammonium bromide (CTAB). As used herein, "passivate" refers to the stabilisation of the gold nanoparticle in solution, before being in contact with the sample. Accordingly, the gold nanoparticle can be stabilised in solution with, for example, citrate ions, such that aggregation of the gold nanoparticles is avoided.

In some embodiments, the first mixture is contacted with AuNPs at a concentration of about 30 pM to about 50 pM. In other embodiments, the AuNPs concentration is about 35 pM to about 50 pM, about 40 pM to about 50 pM, or about 40 pM to about 45 pM.

In some embodiments, the colorimetric indication is obtainable within at least 15 min. In other embodiments, the time is at least 20 min, at least 30 min, at least 40 min, at least 50 min, or at least 60 min.

In some embodiments, a weight ratio of nanoparticles to AuNPs is about 10: 1 to about 600: 1. In other embodiments, the ratio is about 15: 1 to about 580: 1, about 20:1 to about 550: 1, about 20: 1 to about 500: 1, about 20:1 to about 450: 1, about 20:1 to about 400:1, or about 20: 1 to about 350:1. The ratio can, for example, be determined by taking the molar mass of AuNPs to be about 197 g/mol.

This ratio was found to be particularly advantageous in that a lesser amount of AuNPs can be used compared to previous assays and from which analysis can be performed to determine the bacterial cell concentration.

In some embodiments, the assay method further includes a step (c) of quantifying the bacterial cells in the sample after step (b).

Better accuracy in the measurement of the bacterial cell count can be obtained using an analytical instrument. As shown herein, the gold nanoparticles when homogenously dispersed (not aggregated) has a different UV-vis spectra compared to aggregated gold nanoparticles. Without wanting to be bound by theory, the inventors have found that the extent of aggregation of AuNPs can be quantified by measuring the relative peak maxima in the UV-vis spectra. For example, 13 nm AuNPs have a UV-vis peak maxima at about 525 nm. When fully aggregated, the UV-vis peak maxima shifts to about 630 nm. Accordingly, the extent of aggregation can be quantified by following this shift. This can be measured by taking a ratio of the absorbance value at 630 nm relative to the absorbance value at 525 nm. Using this method, the bacterial cell count/concentration in the sample can accordingly be quantified/correlated with the extent of aggregation of AuNPs at a specific duration as the extent of aggregation of gold nanoparticles is directly dependent on the bacterial cell concentration.

In some embodiments, step (c) comprises determining an absorbance ratio of the gold nanoparticle at wavelengths of 630 nm and 525 nm. The ratio can be compared with a calibration plot. The bacterial cell can thus be quantified. The above examples uses 13 nm AuNPs as the colorimetric indicator. If other types of colorimetric nanoparticles are used, the ratio will vary accordingly. The assay can have a detection limit of at least 1 CFU/mL of bacterial cells. In other embodiments, when SPdots are used, the detection limit is about 1 x 10⁵ CFU/mL. In other embodiments, when TPdots are used, the detection limit is about 1 x 10⁴ CFU/mL. In other embodiments, when TPMdots are used, the detection limit is about 1 CFU/mL. It is believed that the detection limit is determined by the synergistic effect of multiple factors, including the concentration of biodots, its surface structure and charge type/density, its interaction with bacterial cell and citrate-protected AuNPs, and the duration of incubating the biodots with bacteria sample. In this regard, an optimal concentrations of biodots to be used can be experimentally determined, as shown in the present invention of SPdots, TPdots, TPMdots for E. coli and S. Aureus. Even while the optimised concentrations for TPdots and TPMdots falls within the same range, the detectable range are different, which may be due to the different surface structure, charge and charge density imparted by the carbohydrate (mannose). Accordingly, by using different carbohydrates, the detectable range can be tuned.

In conclusion, the present invention relates to:

1. A method of detecting bacteria cells in solution based on colour change of gold nanoparticles (AuNPs) probe induced by biogenic nanodots (biodots).

2. The biodots possess unique interactions with bacteria cells (surface binding and cellular uptake) and AuNPs.

3. This method involves the pre-incubation of bacteria sample with biodots, followed by the addition of citrate protected gold nanoparticles for colorimetric detection.

4. This method requires an optimized concentration of biodots and sufficient incubation time (15 mins) to allow interactions of biodots with bacteria sample prior to the addition of gold nanoparticles.

5. A simple "Yes/No" colorimetric method to detect E coli with a detection limit down to 1 CFU/mL: Yes (red color) and No bacteria (blue color) Further, the assay design involves the combination use of unmodified AuNPs and a nanodots (biodots) as reagents for bacteria sensing. The biodots synthesized for the assay possess unique surface properties and interaction with the bacteria cells (tested with E.Coli). The assay principle is based on the measurement of the excess biodots (those that do not interact with bacteria cells) which can cause the color change of AuNPs (red to blue) to determine the lower concentration of bacteria (reverse sensitivity).

The present invention can be used in processing, preparing and manufacturing foods safely. The importance of diligent cleaning, disinfecting and sanitizing in these processes cannot be undermined. To this end, the nanoparticles and/or assay of the present invention can, for example, be used to identify, eliminate or control food-borne pathogens. Further, large scale production of food is an ongoing challenge in that many variable factors come into play for ensuring quality and food safety. Of note, contamination due to bacteria is of importance as a slight or minor contamination can void the whole batch, resulting in large wastage and monetary losses. To this end, the nanoparticles and/or assay of the present invention can, for example, be used to troubleshoot for entry or contamination points.

The present invention can also be used for monitoring pathogenic bacteria in aquacultures and detecting microbial contamination in cosmetic and pharmaceutical products. In this regard, a sample can be obtained and can be concentrated (if required) for quantification.

Examples

Working principle of the colorimetric assay for bacteria detection

The colorimetric bacteria sensor is designed by coupling the unique interactions of biodots with bacteria and with gold nanoparticles (AuNPs). It was found that biodots are able to induce the aggregation of AuNPs, resulting in solution colour change from red (well-dispersed AuNPs) to blue (aggregated AuNPs) (Figure 3-la). On the other hand, biodots were found to interact with bacteria cells as well, possibly via surface interaction and/or being uptaken by bacteria, which is similar to the case that antimicrobial agents are uptaken by bacteria. By purposely mixing specific amount of biodots with bacteria of a relatively high concentration, all these biodots will be "sequestered" by bacteria. AuNPs added subsequently will not aggregate any more due to lacking of excess or free biodots, thus retaining the characteristic wine red colour of AuNPs (Figure 3-lc). When in the presence of bacteria of lesser amount, only partial biodots will be "sequestered" by bacteria, leaving the freely available biodots still interact and cause the AuNPs to aggregate in a lesser extent. As a result, a purple colour indicating partial aggregation of AuNPs was observed (Figure 3-lb). A unique colorimetric assay for detecting bacteria cells in solution is therefore established by pre-incubating biodots with E. coli sample, followed by adding the 13 nm citrate-protected AuNPs as the optical probe. The colour change of the mixture can be observed by naked eyes or examined by UV-vis spectroscopy. A UV-vis absorption spectrum corresponding to the colour change can be obtained in the latter case and the ratio of absorbance at 630 nm and 525 nm (A630/A525) can be employed to evaluate the extent of aggregation of AuNPs (Figure 3-2). The ratio of UV-vis absorption peak is proportional the ratio of aggregated AuNPs and well-dispersed AuNPs respectively, and can provide an indication of the extent of aggregation and hence the amount of bacterial cells present. While a 630 nm and 525 nm wavelength is used in the present disclosure, it would be understood that these wavelengths can shift by about ±5 nm, as the detection of such absorption peak can be influenced by the different equipment used. In addition, if nanoparticles of different sizes are used or if different ligands are used, the absorption peaks may shift as well. By plotting this ratio against E. coli of known concentrations, it is possible that the bacteria can be quantified by referring to the colour change of the mixture solution.

Validation of the colorimetric assay with different design of biodots

In this study, the colorimetric assay was validated using different biodots, namely Ser- PEI (SP) dots, Thr-PEI (TP) dots, and Thr-PEI-mannose (TPM) dots, respectively.

The synthesis route for Threonine-PEI-mannose (TPM) dots follows a two-step hydrothermal process. The amino acid, threonine is first mixed with branched PEI at high temperature and pressure to obtain threonine-PEI (TP) dots. After ultrafiltration, the purified TPdots (<3kD) are then mixed with mannose for a second step heating process (60°C, 48h) to obtain the final TPMdots. The synthesis of SP dots follow a similar route.

The ultra-small TPdots and TPMdots (l-5nm) may allow its uptake into the bacterial cells. In addition, TPMdots show slightly positive zeta potential (~ +7mV) that can possibly enhance initial interactions of TPMdots with bacterial membrane. TPMdots show 98.7% PL remaining after 30 min UV irradiation, thereby indicating that it is sufficiently photo-stable.

TPdots has the lowest minimum inhibitory concentration (MIC) values for both gramnegative E.coli (31 pg/mL) and gram-positive S. aureus (62 pg/mL) after 24 h incubation. In summary, the order of MIC value is TP>SP>TPMdots for E.coli and TP>TPM>SPdots for S. aureus.

For each biodots applied, parameters such as the amount of biodots and the duration of biodots interacting with bacteria prior to addition of AuNPs were studied in order to establish the optimal conditions for bacteria detection. Figure 4 shows the results obtained with SPdots. It can be seen that for a given duration of biodots interacting with bacteria (e.g., 15 min, A), AuNPs do not aggregate at lower concentration of SPdots (<0.239 pg/mL). On the other hand, AuNPs aggregate severely at high concentrations of SPdots (>3.58 pg/mL). A630/A525 overlaps each other and cannot differentiate the bacteria samples with the blank. Only at medium concentrations of SPdots (e.g., 2.69 or 3.07 pg/mL), it is possible to distinguish the bacteria samples from the blank. Similarly, the duration of biodots interacting with bacteria was studied with varied SPdots concentrations and a similar sweet zone of SPdots concentration was also observed (B and C). Finally, the optimal conditions for SPdots were identified as 3.58 pg/mL of SPdots with 30 min of incubation time of SPdots with E coli (D). A linear correlation of E coli concentration with the AuNPs aggregation profile was also established for E coli with concentration of 10⁵ -10⁸ CFU/mL. Separately, the detection limit obtained using SPdots is 10⁵ CFU/mL, which means that it is difficult to differentiate this amount of bacteria from the blank.

Assay results obtained with TPdots and TPMdots are shown in Figure 5 and Figure 6, respectively. Similar to SPdots, a moderate concentration of biodots is needed to differentiate bacteria samples according to their concentration and from the blank. The optimal concentrations were found to be 0.179 pg/mL for both TPdots and TPMdots. In addition, a sufficient duration of biodots interacting with bacteria samples prior to addition of AuNPs is necessary to better differentiate the bacteria samples. The optimal incubation time is 45 and 15 min for TPdots and TPMdots, respectively. A generally long incubation time suggests up-taking of biodots by bacteria as a plausible means of interaction. Interestingly, in the case of TPMdots, the aggregation extent (i.e., A630/A525) of bacteria samples is much lower as compared to the blank sample even for bacteria sample lower to 10⁴ CFU/mL, suggesting a lower limit of detection for assays using TPMdots. The lesser extent of aggregation is in well accordance with the fact that TPMdots are encoded with mannose moiety which binds to cell membrane of E coli, facilitating a stronger and faster interaction of biodots with bacteria thus a higher efficiency in preventing the biodots from inducing the aggregation of AuNPs.

Application of colorimetric "Yes/No" assay for ultrasensitive detection of bacteria

The assay with TPMdots was further applied for bacteria detection at low concentration range of bacteria cells (1 to 1000 CFU/mL). Four concentrations of TPMdots (ranging from 0.167 to 0.182 pg/mL) were tested to validate the working principles of our colorimetric assay for ultrasensitive detection of bacteria (Figure 7 B-E). Due to the obvious colour difference as seen in Figure 7A (colour photograph of assay results with and without bacteria), the assay can serve as a simple 'yes'/'no' screening method for detecting ultralow concentration of bacteria down to 1 CFU/mL. The inventors are not aware of other optical sensors that can achieve such a low limit of detection. More importantly, current assay required no specialized equipment or well-trained operators for accurate detection due to the distinct colour change in response to the presence of bacteria which is obvious and evident enough to be observed by naked eyes and hence easily detected (Figure 7k). In addition to its sensitivity, the current colorimetric assay is also promising for its fast assay time (~15 min) suitable for onsite detection and high- through screening (food safety analysis) application in the factory.

Applications of the present invention can be in food and beverage security, aquatic system monitoring, clinical sample analysis and pharmaceutical and cosmetic product quality.

It will be appreciated that many further modifications and permutations of various aspects of the described embodiments are possible. Accordingly, the described aspects are intended to embrace all such alterations, modifications, and variations that fall within the spirit and scope of the appended claims.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims

1. A colorimetric assay for detecting bacterial cells in a sample, including the steps of: a) contacting the sample with nanoparticles to form a first mixture; and b) subsequently contacting the first mixture with gold nanoparticles, the gold nanoparticles for providing a colorimetric indication; wherein the nanoparticles (from step (a)) are characterised by an amorphous core, the core formed from threonine and polyethylenimine (PEI); wherein a red coloration after step (b) is indicative of the presence of bacterial cells in the sample; and wherein a blue or purple coloration after step (b) is indicative of the absence of bacterial cells in the sample.

2. The colorimetric assay according to claim 1, wherein the nanoparticle is further characterised by a surface, the surface functionalised with at least a carbohydrate.

3. The colorimetric assay according to claim 1 or 2, wherein the carbohydrate is mannose.

4. The colorimetric assay according to any one of claims 1 to 3, wherein the PEI in the nanoparticle core is a branched PEI.

5. The colorimetric assay according to any one of claims 1 to 4, wherein the amorphous core of the nanoparticle has a molecular weight of less than 3 kDa.

6. The colorimetric assay according to any one of claims 1 to 5, wherein the nanoparticle has a mean diameter of about 1 nm to about 8 nm.

7. The colorimetric assay according to any one of claims 1 to 6, wherein the nanoparticle has a zeta potential of more than about +5 mV.

8. The colorimetric assay according to any one of claims 1 to 7, wherein the nanoparticle has a minimum inhibitory concentration (MIC) value against bacterial cells of more than 30 pg/mL.

9. The colorimetric assay according to any one of claims 1 to 8, wherein step (a) is performed for a period of at least 10 min.

10. The colorimetric assay according to any one of claims 1 to 9, wherein step (a) is performed at a temperature of about 15 °C to about 35 °C.

11. The colorimetric assay according to any one of claims 1 to 10, wherein the nanoparticle is provided at a concentration of at least 0.15 pg/mL.

12. The colorimetric assay according to any one of claims 1 to 11, wherein the gold nanoparticle has a mean particle size of about 10 nm to about 20 nm.

13. The colorimetric assay according to any one of claims 1 to 12, wherein the gold nanoparticle is passivated with citrate ions.

14. The colorimetric assay according to any one of claims 1 to 13, wherein the colorimetric indication is obtainable within at least 15 min.

15. The colorimetric assay according to any one of claims 1 to 14, further including a step (c) of quantifying the bacterial cells in the sample after step (b).

16. The colorimetric assay according to claim 15, wherein step (c) comprises determining an absorbance ratio of the gold nanoparticle at wavelengths of 630 nm and 525 nm, and comparing the absorbance ratio with a calibration plot.

17. The colorimetric assay according to any one of claims 1 to 16, having a detection limit of at least 1 CFU/mL of bacterial cells.

18. The colorimetric assay according to any one of claims 1 to 17, wherein a weight ratio of nanoparticles to gold nanoparticles is about 10:1 to about 600: 1.