Saliva sampling devices

Abstract

The present invention relates to saliva sampling devices comprising gellan gum, clay and a crosslinking agent, obtaining them by means of 3D printing, and their use in the field of clinical and toxicological diagnostics.

Description

[0001] SALIVA SAMPLING DEVICES

[0002] The present invention relates to saliva sampling devices comprising gellan gum, clay and a crosslinking agent, obtaining them by means of 3D printing, and their use in the field of clinical and toxicological diagnostics.

[0003] Background

[0004] In the field of clinical and toxicological diagnostics, saliva has been established as a reference matrix, thanks to its peculiar characteristics, in terms of representativeness and ease of collection. For example, saliva is now the most widely used matrix for toxicological controls on the road and numerous applications have been validated in the field of diagnostics that are based on the research of specific salivary biomarkers for the early diagnosis of different diseases. In all these applications, the saliva is generally collected using disposable swabs to be inserted into the oral cavity.

[0005] However, the sample collection is a rather critical phase in the entire analytical protocol and medical waste is also produced in this phase, since the sampling devices that are on the market generally consist of plastic material (e.g. Polyester). The plastic collection supports, such as cellulosics that are widely available on the market, can interfere with the measurement of lipophilic substances such as cannabinoids, as the latter tend to adhere to said supports. After sampling, the biomarkers of interest must be recovered from the device through appropriate methods and solvents, however, not infrequently the extraction can be incomplete or non-reproducible. There is therefore a need to develop saliva sampling devices that can overcome the problems of the existing ones.

[0006] Background Art

[0007] Gellan gum is known in the art for its properties in the field of drug release. In particular, compositions comprising gellan gum, laponite with [1] or without [2] CaCh have been described for this property. Among the most widely used saliva sampling devices already available on the market [3], the following should be highlighted:

[0008] ■ Quanti-Sal: manufactured by Immunalysis, it consists of an absorbent swab made of cellulose with a polypropylene stem and plastic tube, containing a non-azidic buffer solution of about 3 mL. The collection buffer has an indicator for the volume collected, equal to 1 mL±10%.Sampling is carried out by placing the device under the tongue and there is a collection tube for transport [4], It is the most used device for the collection of oral fluid in the forensic toxicology field.

[0009] ■ Statsure: device consisting of an absorbent swab made of cellulose with a volume adequacy indicator and a plastic tube containing a buffer solution. The declared volume of oral fluid collected is equal to ImL. Sample collection is carried out by placing the swab under the tongue.

[0010] ■ Saletto: marketed by the company Porex, it is a device for collecting saliva with a filtering function to reduce the viscosity of saliva samples. The absorbent material is made of a synthetic fibre, there is a colorimetric indicator that indicates having reached the required volume, between 0.5 and 1 mL. The configuration of the Saletto allows to avoid steps such as centrifugation and there are no particular recommendations for transporting the sample. [5] ■ SalivaBio Oral Swab (SOS) is marketed by the company Salimetrics; the absorbent material consists of a synthetic fibre, there is no colorimetric indicator for reaching the sample volume which can have a maximum value of 2 mL. The material is non-toxic, centrifugation at 1500 g for 15 minutes is required and the use of a containment tube is recommended for transport. It is used to search for the following biomarkers: alpha amylase, C-reactive protein, cortisol, cotinine, osteocalcin, testosterone, uric acid [6]

[0011] ■ Salivette, from the company Sarstedt, the absorbent material is cotton, the sampling volume is between 0.8 and 1.4 mL. A centrifugation step at 1000 g for 2 minutes is recommended for the extraction of saliva from the buffer, and containment tubes are used for transport. Unlike other devices, which sample oral fluid in particular positions of the oral cavity, this is used as a chewing gum. [7]

[0012] ■ Versi-Sal: the absorbent material is non-cellulosic, it is inserted under the tongue, the sampling volume is 1.2- 1.4 mL and there is a colorimetric indicator. The use of 2mL Eppendorf is recommended for transport. [8]

[0013] ■ Accu-Sal: device containing an absorbent swab, placed under the tongue for sample collection.

[0014] There is a colorimetric indicator for reaching the recommended volume (1.0-1.1 mL) and a buffer for extraction and stability. [9]

[0015] ■ Oral-Eze: device from Thermo Scientific™, the collection system consists of an absorbent cotton swab and a plastic shield mounted on a plastic handle and a collection tube for stabilising and transporting the sample. Sampling takes place between the lower gum and the cheek withthe plastic protection facing outwards. There is a colorimetric indicator that turns blue when the volume collected is adequate and a sodium azide buffer solution.

[0016] ■ Intercept i2 from OraSure Technologies: the device consists of cotton fibres attached to a nylon stick and a preservative solution in a plastic container. The buffer is impregnated with 3.5% sodium chloride and gelatin. The declared amount of preservative solution is 0.8 mL. The volume of oral fluid collected is equal to ImL, and there is a need, after collection, to centrifuge at 3000 rpm for 5 minutes. [4]

[0017] Other devices described in the literature include the following:

[0018] ■ EP3568687 - Kit consisting of a manifold consisting of dissolvable cellulose acetate nanofibre material and a sample recovery liquid in which the material is dissolvable. The material consists of cellulose acetate which is transformed into nanofibres through electrospinning. The complete dissolution of the material is carried out by treatment with a buffer containing guanidinium isothiocyanate (GITC). In the extraction protocols, the amount of cellulose acetate must not exceed 50 mg of material. The cellulose nanofibres can be made in the form of a nonwoven mass and / or yarns and / or filaments of nanofibres woven together or with other yarns or fibres other than cellulose acetate. To prepare the cellulose acetate nanofibres, the preparation begins with the acetate ester of cellulose; it is derived from cellulose by deconstructing the wood pulp and reacting it with acetic acid and acetic anhydride in the presence of sulphuric acid. It is then subjected to a controlled partial hydrolysis to remove the sulphate group and the acetate groups. The swabs can be produced for the collection of samples from a patient or for the collection of forensic specimens from various surfaces.

[0019] ■ US4580577: Discloses sampling devices using cellulose sponges or absorbent thermoplastic foam from which saliva is mechanically extruded after being absorbed.

[0020] ■ US5260031: Collection system consisting of a nitrocellulose pad with a volume indicator. The collected saliva is about ImL and is extracted using a buffer and mechanical filtration. The disadvantage of the device is that it takes 40 minutes to obtain a sufficient volume of samples from individuals with dry mouth. It cannot be used for road tests.

[0021] ■ US20210205805A1: discloses devices from a porous polymeric matrix that is obtained through covalent chemical crosslinking that does not make it readily dissolvable and biodegradable.

[0022] ■ US20230389900: discloses devices consisting of biodegradable polymeric nanofibres obtained by means of electrospinning processes. However, the matrix requires further processing to increase the hydrophilicity characteristics thereof and therefore the sampling capacity ofbiological fluids.

[0023] ■ W02013123500: discloses devices based on calcium ion cross-linked alginate. The device is dissolvable in specific buffers, however the solutions obtained have high viscosity which interferes with the further handling and analysis of the sample.

[0024] Brief description of the figures

[0025] Figure 1: Saliva sampling device according to the invention obtained by 3D printing before (A) and after (B) lyophilization.

[0026] Figure 2: Comparison between FT-IR spectra obtained from saliva, water, and solutions obtained from dissolving devices of the invention after absorption of saliva.

[0027] Description of the Invention

[0028] The inventors have determined that saliva sampling devices comprising gellan gum, clay and a crosslinking agent allow the recovery of the analytes by simple dissolution of the device in water, thus reducing the probability of analyte retention phenomena on the sampling device, and improving the accuracy of the analytical results. In fact, the incomplete recovery of the analytes of interest from the material used for sampling is a cause of analytical errors, as it leads to an underestimation of the amount of biomarkers actually present in the sample. This phenomenon is observed more with lipophilic substances, for example, among abused drugs including cannabinoids, among endogenous metabolites, lipid biomarkers.

[0029] These materials, being biocompatible, also constitute an advantage from an environmental point of view. Such devices can be produced by means of 3D printing, which is known for its cost-effectiveness, versatility and environmental sustainability.

[0030] In a first aspect, this invention therefore relates to a saliva sampling device comprising gellan gum, clay and a crosslinking agent.

[0031] In an embodiment, said device can be obtained by a process comprising:

[0032] a) obtaining a shape suitable for saliva sampling with an aqueous mixture comprising between 2% and 4% w / v of gellan gum and between 0.1% and 2.0% w / v of clay;

[0033] b) treating the shape obtained in a) with a solution comprising a crosslinking agent; andc) drying the shape obtained in b), so as to obtain a device having between 0.1 and 1.50% w / w of cross-linking agent.

[0034] In an embodiment, the drying is carried out by lyophilization.

[0035] In an embodiment, the aqueous mixture comprises a w / v percentage of gellan gum selected from between 2% and 3.75%, between 2% and 3.5%, between 2% and 3.25%, between 2.25% and 3.75%, between 2.25% and 3.5%, between 2.25% and 3.25%, between 2.5% and 3.75%, between 2.5% and 3.5%, between 2.5% and 3.25%, between 2.75% and 3.75%, between 2.75% and 3.5%, between 2.75% and 3.25%.

[0036] In an embodiment, the aqueous mixture comprises a percentage of gellan gum of 3% w / v.

[0037] In an embodiment, the aqueous mixture comprises a w / v percentage of clay selected from between 0,1% and 1,75%, between 0.1% and 1.5%, from between 0.1% and 1.25%., between 0.1% and 1%, between 0.1% and 0.75%, between 0.15% and 2.0%, between 0.15% and 1.75%, between 0.15% and 1.5%, between 0.15% and 1.25%., between 0.15% and 1%, between 0.15% and 0.75%, between 0.20% and 2.0%, between 0.20% and 1.75%, between 0.20% and 1.5%, between 0.20% and 1.25%, between 0.20% and 1%, between 0.20% and 0.75%, between 0.25% and 2.0%, between 0.25% and 1.75%, between 0.25% and 1.5%, between 0.25% and 1.25%., between 0.25% and 1%, between 0.25% and 0.75%.

[0038] In an embodiment, the aqueous mixture comprises a percentage of clay of 0.5% w / v.

[0039] In an embodiment, the percentage w / w of crosslinking agent in the device obtained after drying is selected from between 0.1% and 1.25%, between 0.1% and 1%, between 0.1% and 0.75%, between 0.2% and 1.5%, between 0.2% and 1.25%, between 0.2% and 1%, between 0.2% and 0.75%, between 0.25% and 1.5%, between 0.25% and 1.25%, between 0.25% and 1% . between 0.25% and 0.75%. In an embodiment, the percentage w / w in crosslinking agent in the device obtained after drying is 0.5%. In an embodiment, the shape is obtained by 3D printing.

[0040] In an embodiment, the clay is a synthetic clay.

[0041] In an embodiment, the synthetic clay is selected from the list comprising laponite, bentonite, montmorillonite and diosmectite.

[0042] In an embodiment, the laponite is laponite XLG.

[0043] In an embodiment, the crosslinking agent is a monovalent or polyvalent ion salt.

[0044] In an embodiment, the monovalent ion is selected from the list comprising Li+, Na+, K+, Ag+and Au+.In an embodiment, the polyvalent ion is selected from the list comprising Ca+2; Mg+2; Fe+2; Fe+3and Zn+2. In an embodiment, the cross-linking agent is calcium chloride.

[0045] In a second aspect, the invention relates to the use of the device according to the first aspect for the qualitative and / or quantitative analysis of one or more analytes present in saliva.

[0046] In an embodiment, the analytes are selected from the list comprising oxidative stress markers, organic and / or inorganic firearms firing residues, psychoactive substances, and cannabinoids.

[0047] In an embodiment, the markers of oxidative stress are isoprostanes.

[0048] Psychoactive substances are described in the 1961 Single Convention on Narcotic Drugs of the United Nations or in the 1971 Convention on Psychotropic Substances of the United Nations, or renamed as New Psychoactive Substance (NPS) by the United Nations Office on Drugs and Crime.

[0049] In an embodiment, the cannabinoids are selected from the list comprising (-) -trans-^9-tetrahydrocannabinol, (THC), cannabinol (CBN), (-) -cannabidiol (CBD), and (±) 11 -nor-9-carboxy-tetrahydrocannabinol.

[0050] All embodiments can be combined with each other.

[0051] The invention will now be further described by the following examples.

[0052] Examples

[0053] Example 1-Inks and related quality of saliva sampling devices obtained by means of 3D printing Ink preparation involves solubilizing gellan gum and laponite XLG in distilled H2O for 5h at 80°C. At the end of the solubilization, the polymer solution obtained is poured into the print cartridge and left overnight at 4°C before being used in the actual printing step.

[0054] The 3D printer used is Cellink's BioX.

[0055] The table below (Tab. 1) shows the composition of all the formulated inks, their printability, the printing conditions used, the resolution and stability of the post-printing sample and finally the stability of the device after the lyophilization process, i.e. the final device, ready for use. For some examples, the amount (in % w / w) in gellan gum, laponite and cross-linking agent after drying is also reported.

[0056] All the samples were printed using 27G needle and the printing parameters indicated in the table were optimized based on that choice.

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[0073] Example 2- Saliva sampling device

[0074] The device of Fig. 1 having dimensions of 17x7x6 mm and holes with a diameter of 3 mm was obtained with the method described in the last row of Tab. 1.

[0075] The presence of holes is useful for increasing the surface of the swab and thus increasing both the volume of saliva that can be collected and the dissolution speed.

[0076] This device allows sampling 250 pL (±10%) of saliva.Example 3- Cannabinoid analysis

[0077] Sampling and dissolution procedure

[0078] The device of Example 2 is inserted into the subject's mouth for a time varying between 10-15 seconds. It is then moved to a plastic tube containing 5 mM of milli-Q water with 0.005 M EDTA heated to 37°C at pH 7 and 500 pl mL of acetonitrile acidified to 0.05% with formic acid.

[0079] This is followed by dissolution in an ultrasonic bath at a temperature suitable for storing the desired analyte (in the case of the cannabinoids CBN, THC and CBD, for example, at a temperature of 35°C it was observed that all the analytes are stable, while passing to 50°C and then to 60°C a reduced stability is observed, especially for THC) until the device is completely dissolved, centrifugation for 10 minutes at a speed of 4500 rpm and collection of the supernatant for analysis.

[0080] Analysis

[0081] After saliva absorption, a device of Example 2 was added with standard mixtures of cannabinoids (THC, CBD and CBN at a concentration of 125 ppb, THC.COOH ((±) 1 l-nor-9-carboxy -tetrahydrocannabinol) at a concentration of 5 ppb and THC-d3 and THC-COOH-d3 at a concentration of 1 ppb. 500 pL of acetonitrile with 0.05% formic acid + 4.5 mL of H2O with 0.005 M EDTA are added to the buffer thus fortified and left at 35°C for 30 min until completely dissolved. It is then followed bythe dLLME extraction: 200 pL of acetonitrile containing 0.05% of HCOOH + 200 pL of chloroform. The chloroform (about 200 pL) is then collected, followed by the drying under a nitrogen flow (37°C). The residue is taken up with 100 pL of methanol, followed by HPLC-MS / MS analysis.

[0082] The analysis of cannabinoid contents by HPLC-MS / MS of the samples thus obtained perfectly mirrors those of the standard mixtures used.

[0083] Example 4 - Analysis of classic drugs of abuse and new psychoactive substances (NPS) Sampling and dissolution procedure

[0084] The swab is inserted into the subject's mouth for about 10-15 seconds. The device allows sampling 250 pL (±10%) of saliva.

[0085] For solubilization, the device is then placed in a plastic tube containing 2 mL of milli-Q water with 0.005 M EDTA heated to 37°C at pH 11 (by NaHCOs / Na2 CO3 0.01 M carbonate buffer).It is left for about 20 minutes at 37°C in the ultrasonic bath to promote the complete solubilisation of the device. The tube is then placed on the vortex for 10 seconds; a centrifugation phase of 10 minutes at a speed of 4500 rpm and the collection of the supernatant for the extraction of the analytes follow.

[0086] Extraction and analysis

[0087] A single extraction and LC-MS / MS analysis is performed to simultaneously detect the possible presence of the listed substances (divided by chemical / pharmacological class).

[0088] Phenylethylamines: Amphetamine, Methamphetamine, 3,4-Methylenedioxyamphetamine (MDA), 3,4-methylenedioxy-N-ethylphetamine (MDEA), 3,4-methylenedioxymethamphetamine (MDMA), 2-(4-iodo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25-I-NBOMe), 4-bromo-2,5-dimethoxyphenethylamine (2C-B), 2,5-dimethoxyphenethylamine (2C-H), 4-isopropylthio-2,5-dimethoxyphenethylamine (2C-T-4), 4-propylthio-2,5-dimethoxyphenethylamine (2C-T-7) Catinones: Buphedrone, Butylone, Methylone, Mephedrone, 4-Methylethcatinone, Diethylcatinone, Methylenedioxypyrovalerone (MDPV)

[0089] Synthetic Cannabinoids: JWH-200, JWH-250, JWH-122, MAM-2201, UR-144, XLR-11 Opioids: Morphine, 6-monoacetylmorphine, Codeine, Methadone, Buprenorphine, Nor buprenorphine, Fentanyl, Acetylfentanyl, Acrylfentanyl, Alfentanyl, a-methylfentanyl, a-methylthiofentanyl, P-hydroxyfentanyl, Butyrylfentanyl, cis-3 -methylthiofentanyl, cis-3 -methylfentanyl, Fluoro-fentanyl, Furanyl-fentanyl, Norfentanyl, Ocfentanyl, Remifentanyl, Sufentanyl

[0090] Alkaloids: Cocaine, Benzoylecgonine (BEG), Norcocaine

[0091] Arylcyclohexylamines: Ketamine, Methoxetamine

[0092] The deuterated internal standards (Cocaine-d3, BEG-d3, Morphine-d3, Fentanyl-d5, MDPV-d8, Norfentanyl-d5, Buprenorphine-d4, MAM2201-d5, JWH200-d5) are added to the solution in which the device was dissolved to a concentration of 10 ppb. Follows a liquid-liquid extraction assisted by the presence of salts (salting out liquid-liquid extraction -SALLE): 0.4 g of NaCl and 1 mL of acetonitrile are added to the solution and the mixture is centrifuged for 10 minutes at 4500 rpm, to accelerate the separation of the aqueous and organic phases, into which the analytes are transferred. Finally, about 500 pL of organic phase is taken with a syringe, which is placed in a vial for HPLC-MS / MS analysis.Experiments were performed by fortifying nine saliva samples added with standard mixtures containing all the aforementioned analytes so as to have, in triplicate, salivary concentrations of 2.5, 25 and 250 ppb and, following extraction, the HPLC-MS / MS analysis of the samples thus obtained perfectly mirrors the concentrations of the standard mixtures used.

[0093] Example 5 - Absence of contamination

[0094] To evaluate the possible release of substances from the device to the saliva, Fourier Transformed-InfraRed spectroscopy (FT-IR) was used. First, a saliva aliquot of 2mL was collected and placed in a falcon. The device of Example 2 was contacted with the saliva for a set time and then removed. The experiment was carried out twice, with variation of contact times (10s for the first evaluation, 30s for the second evaluation), so as to be able to identify any release even in the case of prolonged contact, and then be dissolved as described in Example 3. As references for the control of signals not attributable to any substances released, both milliQ water and an aliquot of the starting saliva, which was not in contact with the devices, were used. For each sample (saliva-device 10s; saliva-device 30s; saliva reference; water reference) FT-IR spectra were then recorded in Total Attenuated Reflectance mode with diamond crystal. For each sample, 5 pL was taken, contacted with the crystal and 7 spectra were recorded (32 scans per spectrum, resolution 4 cm'1, range: 400-4000 cm'1), which were then averaged over a spectrum representative of the sample rate. This procedure was applied 4 times in total, in order to obtain 4 average spectra representative of 4 different aliquots and evaluate the reproducibility of the measurements. These 4 spectra per type of sample were considered highly reproducible, and were therefore averaged in turn to obtain an average spectrum of the sample. As shown in Fig. 2, the different mean spectra for the four samples were compared to clearly identify the signals attributable to the water and those of the substances present in the saliva. Both spectra obtained by contact at 10 s and at 30 s correspond to that of saliva, while there are no obvious signals attributable to the material constituting the device.REFERENCES

[0095] [1] Adrover et al., Pharmaceutics 2019, 77(4), 187; doi 10.3390 / pharmaceuticsl 1040187

[0096] [2]Pacelli etal., European Polymer Journal, 104, 184-193 (2018): doi 10.1016 / j.eurpolymj.2018.04.034 [3] Lukasz Sobczak et al. Evaluation of swabs from 15 commercially available oral fluid sample collection devices for the analysis of commonly abused substances: doping agents and drugs of abuse. Analyst (2020) 145, 7279-728

[0097] [4] Kaarina tangelet al. Drug Testing in Oral Fluid— Evaluation of Sample Collection Devices. Journal of Analytical Toxicology (2008) 32

[0098]

[0005] https : / / www. porexlifesciences. com / saliva-collection-device /

[0099] [6] https: / / salimetrics.com / collection-method / oral-swab-saliva-collection-device

[0100] [7] M. Costa et al. salivette, a relevant saliva sampling device for sars-cov-2 detection, journal of oral microbiology (2021), 13, 1920226

[0101] [8] https: / / 4saliva.com / versisal /

[0102] [9] https: / / 4saliva.com / accu-sal /

Claims

CLAIMS1. Saliva sampling device comprising gellan gum, clay and a crosslinking agent.

2. Device according to claim 1, obtainable by a process comprising the following steps:a) providing a shape suitable for saliva sampling comprising an aqueous mixture comprising between 2% and 4% w / v gellan gum and between 0.1% and 2.0% w / v clay; b) treating the shape obtained in a) with a solution comprising a crosslinking agent; and c) drying the shape obtained in b), so as to obtain a device having between 0.1 and 1.5% w / w of crosslinking agent.

3. Device according to claim 2, wherein the shape is obtained by 3D printing.

4. Device according to claims 1 to 3, wherein the clay is a synthetic clay.

5. Device according to claim 4, wherein the synthetic clay is a laponite.

6. Device according to claims 1 to 5, wherein the crosslinking agent is a monovalent or polyvalent ion salt.

7. Device according to claim 6, wherein the crosslinking agent is a calcium salt, in particular calcium chloride.

8. Use of the device according to claims 1 to 7 for the qualitative and / or quantitative analysis of one or more analytes present in saliva.

9. Use according to claim 8, wherein the analytes are selected from the list comprising oxidative stress markers, organic and / or inorganic firearms firing residues, psychoactive substances and cannabinoids.

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