WO2016102563A1

WO2016102563A1 - An assay for detecting fungal infections

Info

Publication number: WO2016102563A1
Application number: PCT/EP2015/080971
Authority: WO
Inventors: Furong TIAN
Original assignee: Dublin Institute Of Technology
Priority date: 2014-12-22
Filing date: 2015-12-22
Publication date: 2016-06-30

Abstract

An assay for detecting fungal infection of a sample comprises the steps of incubating the sample in a dilute suspension of colloidal gold having a first colour and detecting a change in colour in the dilute suspension from the first colour to a second colour, in which a colour change is indicative of a fungal infection in the sample.

Description

Title

An assay for detecting fungal infections

Introduction

Many things are prone to fungal infection, including foods and beverages and reagents and media employed in cell culturing. In the medical sphere, fungal infections of human is also a recognised problem - examples of fungal infections of humans include fungal toe infections (onychomycosis), athletes foot, thrush, and ringworm. While these infections can be treated, the treatment tends to be easier when the infection is detected at an early stage. Simple and inexpensive methods of detecting fungal infections are known in the art - the Gold standard of such detection methods is the KOH test [2] , however this test requires use of a microscope and as such is not suitable for use as a "bedside" assay.

It is an object of the invention to provide a sinple and inexpensive test for detecting fungal infections that is suitbale for use as a "bedside" assay.

Statements of Invention

The Applicant has discovered that a dilute suspension of colloidal gold changes colour in the presence of fungi, and that the colour change is detectable to the naked human eye. In the embodiments described below, a dilute suspension of colloidal gold formed from chloroauric acid and a HEPES buffer has a red colour, but that when the suspension comes into contact with fungi, a blue colour develops after a short period of time. In the data presented below, a suspension of colloidal gold having a red colour is described and characterised, and used to detect fungal infections from two samples (cell media and toenails) with a sensitivity of 84% and a specificity of 92-94%.

In a first aspect, the invention provides an assay or method for detecting fungal infection of a sample comprising the steps of incubating the sample in a dilute suspension of colloidal gold having a first colour and detecting a change in colour in the dilute suspension from the first colour to a second colour, in which a colour change is indicative of a fungal infection in the sample. Typically, the dilute suspension of collidal gold has a red colour, and in which the colour change indicative of a fungal infection in a sample is optionally a red to blue colour change.

Preferably, the color change is detectable by the naked eye.

Typically, the dilute suspension of colloidal gold comprises a gold nanoparticle concentration of 0.1 to 0.3mM, and preferably of 0.15 to 0.25mM.

Suitabnly, the dilute suspension of colloidal gold is formed by mixing dilute chloroaunc acid (for example, 0.1 to 0.3 mM) with dilute reducing buffer (for example 0.001 to o.oo3mM). Suitably, 8-12 parts dilute chloroaunc acid is mixed with 1-3 parts dilute reducing buffer. Peferably, 8-12 parts of 0.1 to 0.3 mM chloroauric acid is mixed with 1-3 parts 0.001 to 0.003 mM reducing buffer. Ideally, the gold nanoparticles have an average dimension of not greater than lOOnm (for example 20-100nm). Ideally, the gold nanoparticles have an average dimension of not greater than 80nm (for example, 20-80nm). Ideally, the gold nanoparticles have an average dimension of not greater than 50nm (for example, 20-50nm). Typically, the gold nanoparticles are spherical. The average dimansion of the nanoparticles is determined using scanning electgron microscopy as described below.

The invention also provides an assay or method for quantitative determination of a level of a fungal infection in a sample, comprising the step of carrying out the assay of the invention, and including a step of comparing the second colour with one or more reference colours correlating with different levels of fungal infection to provide a quantitative determination of the level of fungal infection in the sample. Preferably, the step of comparing the second colour to a reference colour employs a colour chart displaying a plurality of colours and associated fungal infection level values. In another embodiment, the step of comparing the second colour to a reference colour employs a machine having a sensor configured to measure the second colour and provide a second colour value, and a processor configured to (a) receive the second colour value from the sensor, (b) compare the second colour value with one or more reference colour values correlating with fungal infection levels, and (c) provide an output of a fungal infection level based on comparison step (b).

The invention also provides a diagnostic reagent for use in detecting the presence of a fungal infection in a sample comprising a dilute suspension of colloidal gold having a red colour and typically formed by providing a mixture comprising dilute chloroauric acid solution and dilte reducing buffer, preferably 8-12 parts 0.1-0.3mM chloroauric acid solution and 1-3 parts 0.001-0.003mM of a reducing buffer.

The invention also provides a kit for detection of fungal infection in a sample comprising: a dilute suspension of colloidal gold having a first colour; a device configured for correlating colour of the colloidal gold suspension with presence, absence, or level of fungal infection; and, optionally, a multiwell plate.

Preferably, the device for correlating colour of the colloidal gold suspension with presence, absence, or level of fungal infection comprises a colour chart displaying a plurality of colours and associated fungal infection level values.

Alternatively, the device for correlating colour of the colloidal gold suspension with presence, absence, or level of fungal infection comprises a machine having a sensor configured to measure the second colour and provide a second colour value, and a processor configured to (a) receive the second colour value from the sensor, (b) compare the second colour value with one or more reference colour values correlating with fungal infection levels, and (c) provide an output of a fungal infection level based on comparison step (b). Brief Description of the Figures

Figure 1 Nanotest solution on individual and batches forms

Figure 2 Morphology of fungi under microscopy. (A), Coomassie stain under a lOOx microscope objective. Hypha of fungi shows a long, branching filamentous structure. (B), using a lOx microscope objective. Figure 3: Scanning Electron Microscopy images of gold nano sphere and nano star. Gold nanospheres (A, scale bar is 150 nm)), Nanostars typically have a central core and 5 or more vertices in a 3 dimensional arrangement (B).

Figure 4: The colour of solution changes are correlated with surface plasma resonance (SPR). (A) first pink line indicates control Nanotest without fungi, blue colour wells indicates there is fungi; (B)SPR of red clour a line and blue colour d line. The spectral profile presents the surface plasma resonates at 530 nm, typical of gold nanospheres (a line in panel B) . An absorbance spectrum shows broad peaked, from 550 nm to 700 nm (d line in panel B).

Figure 5: Raman spectrum of fungi There is not Raman enhancement at distilled water (A), nanostar with distilled water (B) and fungi alone (C). There are high intensity Raman shifts at the sample with nanostar (D).

Detailed Description of the Invention Definitions "Fungal infection" as applied to a sample means the presence of fungi in the sample. While the assay of the invention cannot differentiate pathogenic fungi from nonpathogenic fungi, it can quicky, accurately and inexpensively detect the presence of fungi in a sample. "Sample" can mean any sample, for example a sample derived from humans, animals, foods, beverages, or any products used in industry (for example the diagnostics, pharmaceutical, or food production, comnsumer product industry). In one embodiment, the sample is, or is derived from, human tissue (i.e. skin, hair, nail, saliva, or a swab obtained from a human tissue sample. ). In another embodiment, the sample is, or is derived from, a food or beverage product for human or animal consumption. In one embodiment, the sample may be a carrier that has been brought into contact with the sample, for example a liquid in which the sample has been immersed or stored, or a piece of material which has been used to swab a surface of the sample.

"Incubating the sample" means bringing the sample (or material from the sample) into contact with the diagnostic reagent for a period of time sufficient to allow any fungi present come into contact with the gold nanoparticles present in the reagent. The incubation can take place in a well of a multiwell plate or in an eppindorph tube (the details of which will be known to a person skilled in the art), where the diagnostic reagent and sample are incubated together, or in another embodiment the sample can be material obtained from the sample (contained on a swab or cloth which is brought into contact with the sample) which is brought into contact with the diagnostic reagent. For example, a toenail can be swabbed with a piece of cloth, and then the diagnostic reagent can be poured over the cloth.

"Suspension of colloidal gold" means a liquid suspension of gold nanoparticles. Ideally, the gold nanoparticles have an average dimension of less than lOOnm. Ideally, the gold nanoparticles have an average dimension of less than 80nm. Ideally, the gold nanoparticles have an average dimension of less than 50nm. Typically, the gold nanoparticles are spherical. Ideally, the gold nanoparticles are suspended in a Goods Buffer, for example HEPES, POPSO, MOPS, TAPSO or DIPSO. The suspension of colloidal gold is preferably formed by mixing a chloroauric acid with a buffer having a pH range of 6-9, preferably 7-8.5. Examples of such buffers are Goods Buffers,

"Dilute suspension of colloidal gold" means a suspension of colloidal gold comprising O. lmM to 0.3nM gold nanoparticles, and preferably 0.15mM to 0.25nM gold nanoparticles. "Colour change" generally means a colour change that is detectable to the naked eye. In the embodiment described below, the dilute suspension of colloidal gold has a red colour, which changes to blue when the suspension comes into contact with fungi. "Second colour" means the colour that the diagnostic reagent turns to when it comes into contact with fungi or a fungal infection.

"Detectable to the naked" means that the colour change can be detected unaided by the normal human eye, without the need for magnification means oflight analysis instruments.

"Chloroauric acid" is an inorganic compound with a chemical formula HAuCL₄, and it is provided in various forms, induing trihydrate and tetrahydrate forms. It is commonly used to make colloidal gold suspensions. Dilute chloroauric acid means 0.1-0.5mM, typically 0.1 to 0.3mM chloroauric acid solution.

"Buffer" means a buffer, preferably a reducing buffer, having a pH range of 6-9, preferably 7-8.5. Examples of such buffers are Goods Buffer, for example HEPES, POPSO, MOPS, TAPSO or DIPSO. Dilute buffer means 0.001-0.005mM, preferably 0.001-0.005mM buffer solution.

"A device configured for correlating colour of the colloidal gold suspension with presence, absence, or level of fungal infection" broadly means a device or apparatus that can be employed by a user to correlate the colour of the diagnostic reagent with the fungal infection status of the sample, and determine where or not a fungal infection is present, or quantitatively determine the level of fungal infection. In a simple embodiment, the device could be a colour chart which provides one or more colours that correlate with the presence of a fuingal infection. The colour chart may include a number of colours, graded according to the leve of fungal infection. This, for example, the colours could range from sky blue to navy blue, with navy blue being indicated as a low level of infection and navy blue being indicated as a strong level of infection. In another embodiment, the device is a machine, for example a machine having a sensor configured to measure the second colour and provide a second colour value, and a processor configured to (a) receive the second colour value from the sensor, (b) compare the second colour value with one or more reference colour values correlating with fungal infection levels, and (c) provide an output of a fungal infection level based on comparison step (b). "Sensor configured to measure the second colour" should be understood to mean a device that is capable of determining the second colour, and providing data relating to the sewcond colour to a processor. In one embodiment, the sensor is a spectrophotometer configured to detect wacelength in the 550-700nm range.

Experimental A: Materials Hydrogen tetrachloroaurate(III) trihydrate (HAuC14-4H20) and Costar Clear Polystyrene 96-Well Plates were purchased from Fisher Chemical. Ascorbic acid, AgN03, cetyltrimethylammonium bromide (CTAB), lOnm gold colloid suspensions (6 x 10¹² /mL), HEPES buffer and Coomassie Blue solution were purchased from Sigma Aldrich. Ultrapure deionized water (resistivity greater than 18.0 MQcm^"1) was used for all solution preparations and experiments.

B: Synthesis of Colloidal Gold diagnostic reagent A solution containing gold ions (0.2 mM) in HEPES buffer (2 mM).

The stock solutions of 2 mM chloroaunc acid (Solution A) and 0.1 M HEPES buffer (Sigma-Aldrich) in deionized water (Solution B) were prepared. Solution A (10 μΐ) was added to 96 well plate or 500 μΐ Eppendorf tube and mixed with 2 μΐ HEPES buffer in 88 μΐ distilled water. The colour of solution changed from pale yellow to bright red (indicating the formation of gold nanoparticles) immediately.

C: Characterisation of Colloidal Gold diagnostic reagent The stability of the colloidal gold diagnostic reagent solution on individual and batch forms was tested remained stable over 6 months in room temperature. A Perkin Elmer Lambda 900 UV/VIS/NIR Spectrometer and Zetasizer Nano ZS analyser (Malvern Instruments, Worcestershire, UK) were used to measure the absorbance, hydrodynamic particle size and zeta potentials of the nano particles and to observe the formation and stability of NPs.

The diameter, Zeta Potential and Surface Plasma Resonance of the colloidal gold diagnostic reagent solutions were shown stable (Table 1). The weight of gold is 6.78E- 6 gram at 0.2mM chloroauric acid in 100 μΐ volume.

Table 1. Physico-chemical characteristics of Nanotest

Nanotest Hydrodynamic Zeta _max (nm)

Diameter (nm) Potential

(mv)

Month 0 37.7 -26.1 530

Month 1 35.5 -25.2 529

Month 2 38.1 -26.3 530

Month 3 34.7 -25.7 528

Month 4 35.4 -26.2 529

Month 5 37.2 -25.9 530

Month 6 36.6 -25.5 530

D: Preparation of samples of fungal infection for testing 1. Cell culture media: In order to mimic cell culture contamination, complete culture medium composed of RPMI 1640 supplemented with 10% (v/v) Foetal Bovine Serum (Sigma Aldrich) was maintained in 35 mm cell culture dish ( Trueline) under standard culture conditions. After 72hrs exposure at 37°C, 5% CO2, cell medium was removed and petri dishes were thoroughly washed with PBS. Cell culture medium was removed. Petri dishes were observed under x 10 microscope objective Olympus microscopy. The images of living fungi exhibited typically many long Hypha connected on the core of fungi (Figure 2 B). 2. Toenails: The nails from suspicious fungal infection people were collected from anonymous volunteers in nail salons, foot spar. The nails were washed in the 5 ml distil water for further investigation. A 100 μΐ of the sample was stained with 1:16 diluted coommasie blue solution blue solution. The 500 μΐ mixture solution was loaded in Eppendorf tube and centrifuged with 1000 rpm on SHANDON cytospin 3. The samples were mounted with mounting medium and covered with cover glass. The fungal structures were confirmed under xlOO objective Olympus microscopy (Figure 2 A).

E: Assay for detecting fungal infection in a sample

Different volume of samples, such as, 10 μΐ, 50 μΐ and ΙΟΟμΙ were added into the colloidal gold diagnostic reagent of Section B to optimise sample condition. A red to blue colour was detected in a number of the samples, indicating the presence of fungal infection in those samples. The efficiency of the colloidal gold diagnostic reagent in detection of fungal contamination of cell culture medium is 100% comparing to microscopy observation. Blue colour indicated fungi contamination (from top third to fifth line in the image the 96-well plate figure 4 A). The optimum sample volume is 10 μΐ. The detection with the naked eye seems perfectly suited for detecting analyses in laboratories or public places with fewer resources.

The nails from suspicious fungal infection people were collected in 5 ml distilled water. A 10 μΐ of the sample were added into Nanotest following method 1. The colour changed from bright red to blue (indicating the nanostar formation) in 60 second. Our results show the red colour of intensity attributes the sphere shaped gold nanoparticle. When the fungi met the sphere shaped nanoparticles, the solution turn to be blue. The blue colour indicated there are star shaped gold nano particle forming. It is possible to measure fungi contamination by controlling the colour of the nanoparticle from red to blue (figure 4 A).

The information obtained by Nanotest is summarized in a two-by-two table (Table 2). Table 2

Validity was measured by sensitivity and specificity (Table 3).

Table 3

Test/p value Sensitivity Specificity PPV NPV

Nanotest 84 94 84 94

The gold standard is the best single test that is considered the current preferred method of diagnosing a fungi infection. Any new test needs to be compared against existing method KOH. The current gold standard is KOH for the diagnosis of fungi infection [2] .

Test/p value Sensitivity Specificity PPV NPV

KOH 84 92 71 92

There is not statistic different between our method and existing method.

F: Scanning Electron Microscopy of gold nanoparticles

The samples from Nanotest without fungi and with fungi were deposited onto prewashed silicon substrates and spin coated at a speed of 1000 rpm for 20 seconds for SEM. The samples were dried in air and characterised by Electron Microscopy using a Hitachi SU6600 FESEM instrument at an acceleration voltage of 25 kV. Scanning EM images were taken using the SE detector.

Scanning Electron Microscopy was used to confirm the gold nano particles formation from red to blue colour (SEM images show gold nano particles formation from spheres to star shaped nano particles in figure 3). The gold nano particles in red colour show spherical shaped under SEM. The ones in blue colour show star shaped. Our results showed the red colour of intensity attributes the sphere shaped gold nanoparticle. When the fungi met the sphere shaped nanoparticles, the solution turn to be blue. The blue colour indicated there are star shaped gold nano particle forming.

G: Surface plasma resonance

The colour of solution changes was correlated with surface plasma resonance (SPR). The SPR of solution without fungi was at 530 nm. The SPR of solution with fungi was shifted to high wavelength. There is broad peaked, from 550 nm to 700 nm. In the graph, a line indicated surface plasma resonance (SPR) of red color sphere shaped nano particle and d line indicated the one of blue colour star shaped nano particles (figure 4).

H: Raman spectral with nanostar s to confirm fungal infection

Gold nanostars were prepared in aqueous phase via the surfactant-directed, seed- mediated growth method as described in the literature [1]. Growth solution was prepared by adding 0.20 mL of 0.01 M HAuCl₄:4H₂0 to 4.5 ml of 0.1 M CTAB in a plastic test tube while gently mixing. To this solution, 0.030 mL of 0.01 M AgNO? was added. After mixing, the colour of the solution becomes brownish yellow. Then, 0.032 mL of 0.1 M ascorbic acid was added, resulting in a colourless solution. Finally, 0.01 mL of the 10 nm gold colloid suspension (Sigma Aldrich) was added. After gentle mixing, the solution was kept in a water bath at room temperature.

The spectrum of Mycotoxin could be achieved using Ag NPs and SERS within a few minutes [3]. In order to confirm fungal infection by Raman spectroscopy, the blue- purple colour of gold nanostar has been synthesis for surface Raman enhancement. The surfactant CTAB and ascorbic acid were removed by washing with water. The reaction was stopped after 30 minutes, to limit the particle size to ~150nm and prevent aggregation [4] . Khoury et al. have demonstrated that prolonged reaction time can result in increased size of nanostars [3]. Nanostars were subsequently washed by deionized water three times under centrifugation at 4500 RPM for 15 mins. SERS samples were prepared by mixing 25 μΐ of nanostar solution with 25 μΐ of aqueous solutions of the probe from fungi on CaF2 slide. Raman spectroscopy was performed with a HORIBA Jobin Yvon HR800 spectrometer with either a 300 mW 785 nm diode laser as source. Spectral data was collected using a lOx microscope objective over the range 400-1800 cm^"1 with a 10 sec integration time. The detector used was a 16-bit dynamic range Peltier cooled CCD detector. There is not Raman enhancement at distilled water (Figure 5A), nanostar with distilled water (Figure 5B) and fungi alone (Figure 5C). There are high intensity Raman shifts at the sample with nanostar (Figure 5D). The Raman spectroscopy confirmed fungal infection.

REFERENCES

[l]Esenturk EN, and HightWalker AR (2009) Surface-enhanced Raman scattering spectroscopy via gold nanostars. J. Raman Spectrosc 40: 86-91.

[2]Guarner J and Brandt M E (2011) Histopathologic Diagnosis of Fungal Infections in the 21st Century. Clin Microbiol Rev A 24: 247. [3]Singh D, Ganbold E, Cho E, Cho K, Kim D, Choo I, Kim S, Lee C M, Yang S I , loo S (2014) Detection of the mycotoxin citrinin using silver substrates and Raman spectroscopy. Journal of Hazardous Materials 265, 30 2014, 89-95

[4] Khoury CG, Vo-Dinh T(2008) Gold Nanostars For Surface-Enhanced Raman Scattering: Synthesis, Characterization and Optimization. The journal of physical chemistryC, Nanomaterials and interfaces 112:18849-18859.

Claims

1. An assay for detecting fungal infection of a sample comprising the steps of incubating the sample in a dilute suspension of colloidal gold having a first colour and detecting a change in colour in the dilute suspension from the first colour to a second colour, in which a colour change is indicative of a fungal infection in the sample.

2. An assay as claimed in Claim 1 in which the dilute suspension of collidal gold has a red colour, and in which the colour change indicative of a fungal infection in a sample is a red to blue colour change.

3. An assay as claimed in Claim 1 or 2 in which the dilute suspension of colloidal gold comprises a gold nanoparticle concentration of 0.1 to 0.3mM.

4. An assay as claimed in Claim 3 in which the dilute suspension of colloidal gold comprises a gold nanoparticle concentration of 0.15 to 0.25mM.

5. An assay as claimed in any preceding Claim in which the dilute suspension of colloidal gold is formed by mixing dilute chloroauric acid with dilute reducing buffer.

6. An assay as claimed in any preceding Claim in which the color change is detectable by the naked eye.

7. An assay for quantitative determination of a level of fungal infection in a sample, comprising the step of carrying out the assay of any of Claims 1 to 6, and including a step of comparing the second colour with one or more reference colours correlating with different levels of fungal infection to provide a quantitative determination of the level of fungal infection in the sample.

8. An assay as claimed in Claim 7 in which the step of comparing the second colour to a reference colour employs a colour chart displaying a plurality of colours and associated fungal infection level values.

9. An assay as claimed in Claim 7 in which the step of comparing the second colour to a reference colour employs a machine having a sensor configured to measure the second colour and provide a second colour value, and a processor configured to (a) receive the second colour value from the sensor, (b) compare the second colour value with one or more reference colour values correlating with fungal infection levels, and (c) provide an output of a fungal infection level based on comparison step (b).

10. A diagnostic reagent for use in detecting the presence of a fungal infection in a sample comprising a dilute suspension of colloidal gold having a red colour and formed by providing a mixture comprising 8-12 parts 0.1-0.3mM chloroauric acid solution and 1-3 parts 0.001-0.003mM of a buffer.

11. A diagnostic reagent as claimed in Claim 10 formed by providing a mixture comprising 8-12 parts 0.1-0.3mM chloroauric acid solution and 1-3 parts 0.001- 0.003mM of a buffer.

12. A kit for detection of fungal infection in a sample comprising: a dilute suspension of colloidal gold having a first colour; a device configured for correlating colour of the colloidal gold suspension with presence, absence, or level of fungal infection; and, optionally, a multiwell plate.

13. A kit as claimed in Claim 12 in which the device for correlating colour of the colloidal gold suspension with presence, absence, or level of fungal infection comprises a colour chart displaying a plurality of colours and associated fungal infection level values.

14. A kit as claimed in which the device for correlating colour of the colloidal gold suspension with presence, absence, or level of fungal infection comprises a machine having a sensor configured to measure the second colour and provide a second colour value, and a processor configured to (a) receive the second colour value from the sensor, (b) compare the second colour value with one or more reference colour values correlating with fungal infection levels, and (c) provide an output of a fungal infection level based on comparison step (b).