Title of Invention: A Device for Detection of An Acryloyl Group and A Method Thereof Priority Claim

The present application claims priority to Singapore application No. 10201602557S.

Technical Field

The present invention generally relates to a device for detection of an acryloyl group. The present invention also relates to a method for detection of an acryloyl group using the device.

Background Art

In the recent few years, there have been several food scares that range from the discovery of horsemeat in beef in the United Kingdom to the melamine-tainted milk in China, food safety has emerged as an area of great interest to the public. An essential part of food safety is the surveillance of harmful biological and chemical substances that may arise due to disruption of the cold chain, poor hygiene practices during handling of food, or industrial or agricultural chemicals that may contaminate produce or drinking water at its source. One such compound is acrylamide, a known neurotoxin and carcinogen, which can be introduced into drinking water through the use of polyacrylamide as flocculants in water treatment. While exposure to such exogenous sources of acrylamide can be effectively controlled by careful monitoring and screening of water quality, acrylamide is also produced during food processing methods that involve high heat (>180 °C) such as frying and baking. These Maillard or 'browning' reaction between reducing sugars and amino acids resulted in aromatic compounds that provide food such as potato chips their flavor, but also lead to acrylamide formation.

In response to the risk of exposure, the European Food Safety Authority (EFSA) convened a panel to deliver a scientific opinion on the effects of acrylamide from food sources. The panel concluded that, among other things, acrylamide is "genotoxic and carcinogenic", and potentially "increases the risk of developing cancer in all age groups". In particular, the current margins of exposure (MOEs, i.e. the safety threshold divided by the typical dietary exposure) for acrylamide across dietary surveys and age groups are < 500, far lower than that recommended for other genotoxic and carcinogenic substances (MOE > 10,000), and are thus of particular concern with regard to its neoplastic effects. Hence, various food safety agencies in a number of countries recommend a reduction in the levels of acrylamide in food products where possible.

While the need to monitor acrylamide levels in processed food is clear, existing detection methods are ill-suited for widespread adoption. In general, acrylamide levels are determined by the coventional chromatographic techniques such as high performance liquid chromatography (HPLC) or gas chromatography (GC) followed by detection via mass spectrometry or electron capture. These are sensitive approaches, with a limit of detection of 2.65 ppb and a limit of quantitation of 5 ppb. However, the methods are also tedious and complicated, requiring specialized and costly equipment and can only be performed by trained operators. While testing services are available, they are expensive (> USD$200 per test) and with a typical turnaround time of 1 to 2 weeks. Furthermore, because of its high reactivity, low molecular weight and lack of UV absorption, acrylamide detection is challenging even for these dedicated testing facilities.

The demand for alternative detection approaches has led to the development of electrochemical sensors and immunoassay-based methods. Although electrochemical sensors can be sensitive, they may have limited performance shelf life and may suffer from cross -reactivity issues. On the other hand, antibody-based methods are still labour- intensive and too costly to be practical for widespread testing. A fluorescence strategy utilizing co-polymerization of acrylamide with modified quantum dots (QDs) has also been explored. However, colloidal stability, price, and environmental impact of these semiconductor nanocrystals pose a challenge to the adoption of this approach.

Accordingly, there is a need to provide a device and a method that overcome, or at least ameliorate, one or more disadvantages mentioned above. Some of the advantages may include improved portability, sensitivity and speed, streamlined detection steps, as well as reduced cost for detection. Furthermore, other desirable features and characteristics will become apparent from the subsequent detailed description and the appended claims, taken in conjunction with the accompanying drawings and this background of the disclosure.

Summary of Invention

According to a first aspect, there is provided a device for detecting the presence or absence of an acryloyl group in a sample. The device is a first device coupleable to a second device having a transceiver and a camera for detection of an acryloyl group in a sample, the first device comprising: a compartment for receiving a container comprising the sample and a probe; a first light source configured to activate the probe into a reactive intermediate for reacting with the acryloyl group to produce a pyrazoline cycloadduct; a second light source configured to illuminate the pyrazoline cycloadduct so as to excite fluorescence in the pyrazoline cycloadduct; and a processor connected to the first light source and the second light source for activation thereof, wherein when the first device is coupled to the second device, the processor is configured to receive communication from the transceiver of the second device to switch on the first light source for a first period of time to activate the probe and to thereafter switch on the second light source for a second period of time to excite fluorescence, the first device being aligned with the camera of the second device such that the camera of the second device captures an image of the sample during the second period of time to indicate the presence or absence of the acryloyl group in the sample. According to a second aspect, there is provided a method for detecting the presence or absence of an acryloyl group in a sample, the method comprising: introducing a probe into a sample in a container; inserting the container into a compartment of a first device for detection of an acryloyl group in the sample; switching on a first light source in the first device to illuminate the sample to activate the probe into a reactive intermediate for reacting with the acryloyl group to produce a pyrazoline cycloadduct; switching on a second light source in the first device to illuminate the sample so as to excite fluorescence in the pyrazoline cycloadduct; and capturing an image of the fluorescence in the pyrazoline cycloadduct to determine the presence or absence of the acryloyl group in the sample.

According to a third aspect, there is provided a non-transitory computer readable medium having stored thereon executable instructions for a processor, wherein the processor is controlled to perform steps comprising: communicating instructions to a processor of a first device coupled to a second device comprising the processor for detection of the acryloyl group in a sample to performs steps comprising: switching on a first light source of the first device to illuminate the sample and a probe in a compartment of the first device for a first period of time to activate the probe into a reactive intermediate for reacting with the acryloyl group to produce a pyrazoline cycloadduct; and switching on a second light source of the first device for a second period of time to illuminate the pyrazoline cycloadduct so as to excite fluorescence in the pyrazoline cycloadduct, and activating a camera in the second device and coupled to the processor to capture an image of the sample to determine the presence or absence of the acryloyl group in the sample.

Advantageously, the present device and method leverage the ubiquity of mobile devices, e.g. smart phones, utilising the onboard camera and processing power for fluorescence measurement and analysis, respectively. The performance of the device and method for detection of an acryloyl group in a sample is comparable to that of a research-grade fluorescence plate reader.

Advantageously, the present method can be implemented either in an automated manner through a software application installed on the mobile device to perform the steps as described herein, or in a manual manner that allows users to tweak the operation conditions at one or more of the steps as described herein.

Advantageously, the activation of the camera of the mobile device may also allow measurements to be made using identical settings, resulting in minimal differences between sessions and thus obviating the construction of standard curves with each test.

Further, by utilizing a wireless interface between the phone and the attachment, it is possible for users to quickly modify the device for compatibility with various types of mobile devices with a simple re-design of an interfacing layer where the optical filter and the macro lens can be placed.

Furthermore, with the portability and speed rendered by the present device and method, acryloyl group (e.g. acrylamide) detection can be performed by field inspectors, food producers that includes small businesses, and even health-conscious consumers, who can act as the first-line screening to determine whether certain foods require more thorough testing.

Definitions

The following words and terms used herein shall have the meaning indicated:

In the definitions of a number of substituents below or as described throughout this disclosure, the substituent groups may be a terminal group or a bridging group. This is intended to signify that the use of the term is intended to encompass the situation where the group is a linker between two other portions of the molecule as well as where it is a terminal moiety. Using the term alkyl as an example, some publications would use the term "alkylene" for a bridging group and hence in these other publications there is a distinction between the terms "alkyl" (terminal group) and "alkylene" (bridging group). In the present disclosure, no such distinction is made and most groups may be either a bridging group or a terminal group.

The term "acryloyl group" may refer to a form of enone with structure H ₂ C=CH-C(=0)-. It is also known as prop-2-enoyl, "acrylyl" or simply "acryl". Compounds containing an acryloyl group can be referred to as "acrylic compounds". An acrylic compound is typically an α,β- unsaturated carbonyl compound, containing a carbon-carbon double bond and a carbon-oxygen double bond (carbonyl) separated by a carbon-carbon single bond, thus possessing properties characteristic for both functional groups: at the C=C bond: electrophilic addition of acids and halogens, hydrogenation, hydroxylation and cleavage of the bond; at the C=0 bond: nucleophilic substitution (such as in esters) or nucleophilic addition (such as in ketones). The carboxyl group of acrylic acid can react with ammonia to form acrylamide, or with an alcohol to form an acrylate ester. In addition, since both double bonds are separated by a single C-C bond, the double bonds are conjugated.

In the present application, it is the acryloyl group comprised in a sample that reacts with a reactive intermediate of a probe to produce a fluorescence product (e.g. pyrazoline cycloadduct). Therefore, in view of the present patent specification, it can be appreciated by those skilled in the art that acrylamide, acrylate, acrylic acid, methacrylamide, methyl acrylate, and/or maleic acid monoamide that include an acryloyl group can be detected and quantified using the device and method as described herein.

The phrase "optionally substituted" is to be interpreted broadly to mean that the group to which this term refers to may be unsubstituted, or may be substituted with one or more groups independently selected from, but not limited to, oxygen, sulfur, halogen, alkyl, acyl, ester, amino, amide, carboxylic acid, carbonyl, urea, alkoxy, alkyloxy, alkenyl, alkynyl, sulfonamide, aminosulfonamide, sulfonylurea, oxime, cycloalkyl, aryl, heterocycloalkyl and heteroaryl. Usually these groups have 1 to 12 carbon atoms, if they contain carbon atoms.

The term "halogen" or variants such as "halide" or "halo" as used herein refers to fluorine, chlorine, bromine and iodine or a group 17 element of the periodic table.

The term "alkyl" may refer to a straight-or branched-chain alkyl group having from 1 to 12 carbon atoms or any number of carbon atoms falling within this range in the chain. Exemplary alkyl groups include methyl (Me), ethyl (Et), n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert- butyl (tBu), pentyl, isopentyl, tert-pentyl, hexyl, isohexyl, and the like.

The term "acyl" may mean a -C(0)-R radical, wherein R is an optionally substituted C ₁ -C ₁₂ - alkyl, C ₂ -C ₁₂ -alkenyl, cycloalkyl having 3 to 12 carbon atoms, or aryl having 6 or more carbon atoms, or a 5 to 6 ring membered heterocycloalkyl or heteroaryl group having 1 to 3 hetero atoms select from N, S or O.

The term "ester" includes within its meaning -0-C(0)-alkyl- and -C(0)-0-alkyl- groups.

The term "amino" as used herein may refer to groups of the form -NR ^a R ^b wherein R ^a and R ^b are individually selected from the group including but not limited to hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, and optionally substituted aryl groups. The term "amino" may include an amine group (i.e. -NH ₂ ) or a substituted amine group as defined below.

The term "amide" as used herein may refer to groups of the form -C(0)-NR ^c -alkyl- wherein R ^c is selected from the group including but not limited to hydrogen, optionally substituted alkyl, optionally substituted alkenyl, and optionally substituted aryl groups.

The term "amine" as used herein refers to groups of the form NR ^d R ^e -alkyl- wherein R ^d and R ^e are individually selected from the group including but not limited to hydrogen, optionally substituted alkyl, optionally substituted alkenyl, and optionally substituted aryl groups. The - alkyl- groups in the "amide" and "amine" can be optionally substituted and preferably have 2 to 12 carbon atoms, more preferably 2 to 6 carbon atoms or any number of carbon atoms falling within these ranges.

The term "carboxylic acid" or variants such as "carboxyl" may be intended to refer to a molecule having the group having -C(0)OH.

The term "carbonyl" may refer to a molecule having the group R ^f -C(0)-R ⁸ , wherein R ^f and R ⁸ may be an optionally substituted C ₂ -C ₁₂ -alkenyl, cycloalkyl having 3 to 12 carbon atoms, or aryl having 6 or more carbon atoms, or a 5 to 6 ring membered heterocycloalkyl or heteroaryl group having 1 to 3 hetero atoms select from N, S or O. This term may encompass a ketone.

The term "alkoxy" or variants such as "alkoxide" or "alkyloxy" as used herein may refer to an - O-alkyl radical. Representative examples include, for example, methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy, and the like.

The term "alkenyl group" includes within its meaning divalent ("alkenylene") straight or branched chain unsaturated aliphatic hydrocarbon groups having from 2 to 12 carbon atoms or any number of carbon atoms falling within this range and having at least one double bond, of either E, Z, cis or trans stereochemistry where applicable, anywhere in the alkyl chain. Examples of alkenyl groups include but are not limited to ethenyl, vinyl, allyl, 1 -methyl vinyl, 1- propenyl, 2-propenyl, 2-methyl-l-propenyl, 2-methyl-l-propenyl, 1-butenyl, 2-butenyl, 3- butentyl, 1,3-butadienyl, 1-pentenyl, 2-pententyl, 3-pentenyl, 4-pentenyl, 1,3-pentadienyl, 2,4- pentadienyl, 1 ,4-pentadienyl, 3-methyl -2-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3- hexadienyl, 1 ,4-hexadienyl, 2-methylpentenyl, 1-heptenyl, 2-heptentyl, 3-heptenyl, 1-octenyl, 1- nonenyl, 1-decenyl, and the like.

The term "alkynyl" as used herein, unless otherwise specified, may refer to a branched or unbranched hydrocarbon group of 2 to 12 or any number of carbon atoms falling within this range and containing at least one triple bond, such as acetylenyl, ethynyl, n-propynyl, isopropynyl, n-butynyl, isobutynyl, t-butynyl, octynyl, decynyl and the like.

The term "cycloalkyl" as used herein may refer to a stable non-aromatic monocyclic or poly cyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, which may include fused or bridged ring systems, having from three to fifteen carbon atoms or any number of carbon atoms falling within this range. The "cycloalkyl" may be attached to the rest of the molecule by a single bond. The "cycloalkyl" may be saturated i.e. containing single C-C bonds only. Examples of monocyclic cycloalkyls include, e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. The term "aromatic group", or variants such as "aryl" or "arylene" as used herein may refer to monovalent ("aryl") and divalent ("arylene") single, polynuclear, conjugated and fused residues of aromatic hydrocarbons having from 6 to 12 carbon atoms or any number of carbon atoms falling within this range. Examples of such groups include phenyl, biphenyl, naphthyl, phenanthrenyl, and the like.

The term "heterocycloalkyl" may refer to a saturated monocyclic, bicyclic, or polycyclic ring containing at least one heteroatom selected from nitrogen, sulfur, oxygen, preferably from 1 to 3 heteroatoms in at least one ring. Each ring may be from 3 to 12 membered or having any number of carbon atoms within this range. Examples of heterocycloalkyl substituents include pyrrolidyl, tetrahydrofuryl, tetrahydrothiofuranyl, piperidyl, piperazyl, tetrahydropyranyl, mo hilino, 1,3-diazapane, 1,4-diazapane, 1,4-oxazepane, and 1,4 oxathiapane.

The term "heteroalkyl" refers to a straight-or branched-chain alkyl group having from 2 to 12 atoms in the chain or any number of atoms falling within this range, one or more of which is a heteroatom selected from S, O and N. Exemplary heteroalkyls include alkyl ethers, secondary and tertiary alkyl amines, alkyl sulfides, and the like.

The term "heteroaryl" as used herein may refer to an aromatic monocyclic or multicyclic ring system comprising about 5 to about 12 ring atoms, preferably about 5 to about 10 ring atoms or any number of atoms falling within this range, in which one or more of the ring atoms is an element other than carbon, for example nitrogen, oxygen or sulfur, alone or in combination. The term "heteroaryl" may also include a heteroaryl as defined above fused to an aryl as defined above. Non- limiting examples of suitable heteroaryls include pyridyl, pyrazinyl, furanyl, thienyl, pyrimidinyl, pyridone (including N-substituted pyridones), isoxazolyl, isothiazolyl, oxazolyl, thiazolyl, pyrazolyl, furazanyl, pyrrolyl, pyrazolyl, triazolyl, 1 ,2,4-thiadiazolyl, pyrazinyl, pyridazinyl, quinoxalinyl, phthalazinyl, oxindolyl, imidazo[l,2-a]pyridinyl, imidazo[2,l-b]thiazolyl, benzofurazanyl, indolyl, azaindolyl, benzimidazolyl, benzothienyl, quinolinyl, imidazolyl, thienopyridyl, quinazolinyl, thienopyrimidyl, pyrrolopyridyl, imidazopyridyl, isoquinolinyl, benzoazaindolyl, 1 ,2,4-triazinyl, benzothiazolyl and the like. The term "heteroaryl" also refers to partially saturated heteroaryl moieties such as, for example, tetrahydroisoquinolyl, tetrahydroquinolyl and the like. Heteroaryl groups may be optionally substituted.

The word "substantially" does not exclude "completely" e.g. a composition which is "substantially free" from Y may be completely free from Y. Where necessary, the word "substantially" may be omitted from the definition of the invention.

Unless specified otherwise, the terms "comprising" and "comprise", and grammatical variants thereof, are intended to represent "open" or "inclusive" language such that they include recited elements but also permit inclusion of additional, unrecited elements.

As used herein, the term "about", in the context of concentrations of components of the formulations, typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 3% of the stated value, more typically, +/- 2% of the stated value, even more typically +/- 1% of the stated value, and even more typically +/- 0.5% of the stated value.

Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Certain embodiments may also be described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the embodiments with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, skilled artisans will appreciate that elements in the figures are illustrated for simplicity and clarity and have not necessarily been depicted to scale. For example, the dimensions of some of the elements in the illustrations, block diagrams or flowcharts may be exaggerated in respect to other elements to help to improve understanding of the present embodiments

Brief Description of Drawings

The accompanying drawings illustrate a disclosed embodiment and serves to explain the principles of the disclosed embodiment. It is to be understood, however, that the drawings are designed for purposes of illustration only, and not as a definition of the limits of the invention.

[Fig. 1A] is a reaction scheme between probe 1 and acrylamide.

[Fig. IB] shows the formation of the fluorescence product in the presence of probe 1 and acrylamide.

[Fig. 2A] shows fluorescence images of acrylamide samples after photoactivation with probe 1, at concentrations of 100 nM to 1 μΜ (100 nM increments, top) and 1 to 10 μΜ (1 μΜ increments, bottom).

[Fig. 2B] is a graph showing the fluorescence results of 20 acrylamides samples against the respective acrylamide concentrations.

[Fig. 2C] shows a detection of other acrylates with an acryloyl group using the probe 1.

[Fig. 3A] shows an embodiment of the device for detection of an acryloyl group in a sample.

[Fig. 3B] shows an Apple iPhone 5 to serve as the mobile device to work in conjunction with the device for detection of an acryloyl group in a sample.

[Fig. 3C] shows an example of a receiver of the device for detection of an acryloyl group in a sample. The receiver in this example is a dark chamber.

[Fig. 3D] shows a cross sectional view of the device for detection of an acryloyl group in a sample when attached to the mobile device. [Fig. 4A] shows five fluorescence microscopy images of the device for detection of an acryloyl group that is used for the activation of probe in the presence of acrylamide.

[Fig. 4B] is a graph showing normalized intensity against activation time.

[Fig. 4C] is a graph showing pixel values against acrylamide concentration of the samples. [Fig. 4D] is a graph showing fluorescence curves of various samples that are extracted in accordance with aqueous extraction protocols.

[Fig. 4E] is a graph showing fluorescence curves of various samples that are extracted in accordance with QuEChERS extraction protocols.

[Fig. 4F] is a graph showing fluorescence intensities of reaction products in presence of acetonitrile

[Fig. 5] shows a schematic diagram of acrylamide extraction from food matrices.

[Fig. 6] shows a comparison of acrylamide content detected between the present method and LC-MS/MS performed by a commercial laboratory.

[Fig. 7] shows a paper chromatography that may be employed to separate acrylamide from co-extractants which may react with the probe.

Detailed Description of Embodiments

Embodiments of the present invention will be described, by way of example only, with reference to the drawings. Like reference numerals and characters in the drawings refer to like elements or equivalents.

Some portions of the description which follows may be explicitly or implicitly presented in terms of algorithms and functional or symbolic representations of operations on data within a computer memory. These algorithmic descriptions and functional or symbolic representations are the means used by those skilled in the data processing arts to convey most effectively the substance of their work to others skilled in the art. An algorithm is here, and generally, conceived to be a self-consistent sequence of steps leading to a desired result. The steps are those requiring physical manipulations of physical quantities, such as electrical, magnetic or optical signals capable of being stored, transferred, combined, compared, and otherwise manipulated.

Unless specifically stated otherwise, and as apparent from the following, it will be appreciated that throughout the present specification, discussions utilizing terms such as "activating", "switching on", "analysing", "capturing", "indicating", "providing", "quantification", or the like, refer to the action and processes of a computer system, or similar electronic device, that manipulates and transforms data represented as physical quantities within the computer system into other data similarly represented as physical quantities within the computer system or other information storage, transmission or display devices.

The present specification also discloses apparatus for performing the operations of the methods, e.g. the device for detection of an acryloyl group in a sample. Such apparatus may be specially constructed for the required purposes, as described in an embodiment below, or may comprise a computer or other computing device selectively activated or reconfigured by a computer program stored therein. The algorithms and displays presented herein are not inherently related to any particular computer or other apparatus. Various machines may be used with programs in accordance with the teachings herein. Alternatively, the construction of more specialized apparatus to perform the required method steps may be appropriate. The structure of a computer will appear from the description below. The mobile device may be for example, a mobile terminal such as a smartphone or a tablet with a camera and a mobile operating system, such as Windows of Microsoft, iOS of Apple Inc. or Android of Google Inc. It can be appreciated to those skilled in the art that the mobile device is a form of a computer that a non-transitory computer readable medium can be installed on.

In addition, the present specification also implicitly discloses a computer program, in that it would be apparent to the person skilled in the art that the individual steps of the method described herein may be put into effect by computer code. The computer program is not intended to be limited to any particular programming language and implementation thereof. It will be appreciated that a variety of programming languages and coding thereof may be used to implement the teachings of the disclosure contained herein. Moreover, the computer program is not intended to be limited to any particular control flow. There are many other variants of the computer program, which can use different control flows without departing from the spirit or scope of the invention.

Furthermore, one or more of the steps of the computer program may be performed in parallel rather than sequentially, unless otherwise mentioned. Such a computer program may be stored on any computer readable medium, e.g. a non-transitory computer readable medium. The computer readable medium may include storage devices such as magnetic or optical disks, memory chips, or other storage devices suitable for interfacing with a computer. The computer readable medium may also include a hard-wired medium such as exemplified in the Internet system, or wireless medium such as exemplified in the GSM mobile telephone system. The computer program when loaded and executed on such a general-purpose computer effectively results in an apparatus that implements the steps of the preferred method.

In an embodiment shown in Fig. 3A, The device for detection of an acryloyl group in a sample comprises components including: a compartment for receiving a container comprising the sample and a probe; a first light source configured to activate the probe into a reactive intermediate for reacting with the acryloyl group to produce a pyrazoline cycloadduct; a second light source configured to illuminate the pyrazoline cycloadduct so as to excite fluorescence in the pyrazoline cycloadduct; and a processor connected to the first light source and the second light source for activation thereof.

In the present application, the device for detection of an acryloyl group in a sample is interchangeably referred to as a "first device" to differentiate itself with a second device that has a transceiver and a camera.

In an embodiment, the second device can be a mobile device. As described above, the mobile device may be a mobile terminal such as a smartphone or a tablet with a camera and a mobile operating system, such as Windows of Microsoft, iOS of Apple Inc. or Android of Google Inc. The second device may also have a processor to control the camera thereon. The controlling of the camera may be a manual process or an automated process realised by a software application installed and run on the mobile device.

In the present application, the device is coupleable to the second device. When the device is coupled to the second device, the processor is configured to receive communication from the transceiver of the second device to switch on the first light source for a first period of time to activate the probe and to thereafter switch on the second light source for a second period of time to excite fluorescence. When coupled, the device is aligned with the camera of the second device such that the camera of the second device captures an image of the sample during the second period of time to indicate the presence or absence of the acryloyl group in the sample.

In an embodiment of the device, the processor of the device is configured to establish a wireless connection with the transceiver of the second device to communicate with the second device. The wireless connection can be a Bluetooth connection, a WiFi connection, etc.

In an embodiment of the device, the processor of the device is configured to communicate with the transceiver of the second device to switch the first light source and the second light source in response to one or more instructions from the transceiver of the second device. In the embodiment, a software application may be installed into the second device to initiate the communication of the one or more instructions to the device.

In an embodiment of the device, the first light source comprises one or more ultraviolet B (UVB) light-emitting diodes (LEDs). The second light source comprises one or more ultraviolet A (UVA) light-emitting diodes (LEDs).

In an embodiment when the first light source comprises one UVB LED, the processor may switch on the one or more UVB LEDs for sufficient time to activate the probe, for less than or more than 15 minutes, preferably for 15 minutes or less, to undergo a cycloreversion reaction that results in the reactive intermediate. In this embodiment, the first period of time can be in the range of 1 to 5 minutes, 1 to 10 minutes, or 1 to 15 minutes. For example, the first period of time can be 15 minutes, 14 minutes, 13 minutes, 12 minutes, 11 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute or less than 1 minute.

In an embodiment of the device when the second light source comprises one UVA LED, the processor may switch on the UVA LED for sufficient time to excite fluorescence in the pyrazoline cycloadduct, for less than or more than 1 minute, preferably for about 1 minute.

Those skilled in the art can increase the number of LEDs to further reduce the time required for activation, and increase the number of UBA laser diodes to further reduce the time required for excitation.

The device may further include a power circuit configured to provide power to the processor, the first light source and the second light source. The receiver, the first light source, the second light source and the power circuit can be enclosed within a housing. The housing can further comprise the compartment for receiving the container that comprises the sample and the probe for detection. In an embodiment of the device, the power circuit may include a re-chargeable battery (e.g. a Li-Po battery) for powering the device, a charging circuit configured to power the device off a USB voltage source and connected to the re-chargeable battery and the processor, and a voltage regulator to regulate the output from the processor so as to power the one or more UVB LED. The housing is coupleable to the second device.

The device may further comprise an optical filter. The optical filter can be configured to attenuate a light from the second light source while allowing the fluorescence to be imaged therethrough. When the device is coupled to the second device, the optical filter is aligned with the camera of the second device such that the camera captures the fluorescence of the pyrazoline cycloadduct in the image.

The device may further comprise a macro lens. When the device is coupled to the second device, the macro lens is aligned with the camera of the second device to facilitate focus the sample onto the sample.

In an embodiment of the device, the compartment is configured to receive the container in the form of a cuvette, a microcentrifuge tube, or a dipstick.

In an embodiment, the sample as described above may be any sample containing acryloyl group. The acryloyl group may comprise acrylamide, acrylate, acrylic acid, methacrylamide, methyl acrylate and/or maleic acid monoamide.

In an embodiment, the probe may comprise a diaryltetrazole compound. The diaryltetrazole compound may have the formula (I) :

Formula I

wherein each of Rj to R ₁₀ is independently selected from the group consisting of hydrogen, oxygen, sulfur, hydroxyl, halogen, optionally substituted alkyl, optionally substituted acyl, optionally substituted ester, optionally substituted amino, optionally substituted amine, optionally substituted amide, optionally substituted carboxylic acid, optionally substituted carbonyl, optionally substituted urea, optionally substituted alkoxy, optionally substituted alkyloxy, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted sulfonamide, optionally substituted aminosulfonamide, optionally substituted sulfonylurea, optionally substituted oxime, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocycloalkyl, optionally substituted heteroalkyl, optionally substituted alcohol and optionally substituted heteroaryl. In an embodiment, the diaryltetrazole compound may be

In an embodiment, the reactive intermediate can be a compound comprising a nitrile imine dipole. It may be noted that the reactive intermediate is derived from a photoactivated 1,3- dipolar cycloaddition reaction between a diaryltetrazole and an acryloyl group. Upon photo-irradiation at a particular wavelength, for instance 302 nm, the diaryltetrazole may undergo a cycloreversion reaction to generate a highly reactive nitrile imine dipole with the release of nitrogen. This nitrile imine dipole may subsequently react with the acrylic or acrylate dipolarophile to produce a pyrazoline cycloadduct which may emit fluorescence upon photoactivation. Nitrile imines may be classified as a class of organic compounds sharing a common functional group with the general structure Rx-CN-NRy corresponding to the conjugate base of an amine bonded to the N-terminus of a nitrile. Accordingly, Rx and Ry when used in this context may independently be an optionally substituted organic moiety comprising 1 to 12 carbons or any number of carbon atoms falling within this range. Such an organic moiety may comprise the optional substituents as defined for Rj to R ₁₀ . In an embodiment, when the image of the sample captures the fluorescence, it indicates a detection of the acryloyl group in the sample. That is, when there is fluorescence captured in the image, the acryloyl group is present in the sample. When there is no fluorescence captured in the image, the acryloyl group is not present in the sample.

In a scenario where the acryloyl group is present in the sample, the image of the sample captures the fluorescence. Pixel values of the image of the sample can be analysed for quantification of the acryloyl group in the sample. For example, when the acryloyl group is acrylamide, the volume of the acrylamide comprised in the sample can be determined by the second device by comparison with a standard curve of acrylamide concentration in the same sample. The standard curve can be obtained by acrylamide detection performed by a conventional method on a conventional device, such as LC-MS/MS testing.

The method for detecting the presence or absence of an acryloyl group in a sample comprise steps including: (1) introducing a probe into a sample in a container; (2) inserting the container into a compartment of a first device for detection of an acryloyl group in the sample; (3) switching on a first light source in the first device to illuminate the sample to activate the probe into a reactive intermediate for reacting with the acryloyl group to produce a pyrazoline cycloadduct; (4) switching on a second light source in the first device to illuminate the sample so as to excite fluorescence in the pyrazoline cycloadduct; and (5) capturing an image of the fluorescence in the pyrazoline cycloadduct to determine the presence or absence of the acryloyl group in the sample.

As mentioned above, in the present application, the "first device" is interchangeably used as the "device" for detection of an acryloyl group in a sample.

The method can further comprise a step of coupling the device to a second device having a transceiver and a camera. In step (5), i.e. the step of capturing the image of the fluorescence in the pyrazoline cycloadduct, the method comprises activating the camera of the second device to capture the image of the fluorescence in the pyrazoline cycloadduct.

The method can further comprise a step of establishing a wireless connection between the processor of the device and the transceiver of the second device. In this manner, in steps (4) to (5), the method can further comprise a step of activating a software application stored in the second device to communicate instructions to the processor of the first device to switch on the first light source for a first period of time to activate the probe; and thereafter switch on the second light source for a second period of time to excite fluorescence; and communicate instructions to a processor of the second device to activate the camera of the second device to capture an image of the fluorescence within the second period of time. In an embodiment, the step (5) can be activated by the software application stored in the second device to communicate instructions to a processor of the second device to instruct the camera of the second device to capture the image.

In step (3), the method may comprise switching on the first light source for less than or more than 15 minutes, preferably for 15 minutes or less. Preferably, the method may comprise switching on the first light source for a first period of time in a range of 1 to 5 minutes, 1 to 10 minutes, or 1 to 15 minutes. For example, the first period of time can be 15 minutes, 14 minutes, 13 minutes, 12 minutes, 11 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute or less than 1 minute.

In step (4), the method may comprise switching on the second light source for less than or more than 1 minute, preferably for around 1 minute.

In an embodiment, the step (5) can be performed during the step (4) is performed, i.e. during the 1 minute of the UVA LED excitation. It can be appreciated by the skilled person that the step (5) can also be performed after the step (4) has completed.

In an embodiment of the method, the sample as described above may be any sample containing acryloyl group. The acryloyl group may comprise acrylamide, acrylate, acrylic acid, methacrylamide, methyl acrylate, and/or maleic acid monoamide.

In an embodiment of the method, the probe comprises a diaryltetrazole compound. The diaryltetrazole compound may have the formula (I):

Formula I

wherein each of Rj to R ₁₀ is independently selected from the group consisting of hydrogen, oxygen, sulfur, hydroxyl, halogen, optionally substituted alkyl, optionally substituted acyl, optionally substituted ester, optionally substituted amino, optionally substituted amine, optionally substituted amide, optionally substituted carboxylic acid, optionally substituted carbonyl, optionally substituted urea, optionally substituted alkoxy, optionally substituted alkyloxy, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted sulfonamide, optionally substituted aminosulfonamide, optionally substituted sulfonylurea, optionally substituted oxime, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocycloalkyl, optionally substituted heteroalkyl, optionally substituted alcohol and optionally substituted heteroaryl. In an embodiment, the diaryltetrazole compound may be

In an embodiment of the method, the reactive intermediate is a compound comprising a nitrile imine dipole. It may be noted that the reactive intermediate is derived from a photoactivated 1,3-dipolar cycloaddition reaction between a diaryltetrazole and an acryloyl group. Upon photo-irradiation at a particular wavelength, for instance 302 nm, the diaryltetrazole may undergo a cycloreversion reaction to generate a highly reactive nitrile imine dipole with the release of nitrogen. This nitrile imine dipole may subsequently react with the acrylic or acrylate dipolarophile to produce a pyrazoline cycloadduct which may emit fluorescence upon photoactivation. Nitrile imines may be classified as a class of organic compounds sharing a common functional group with the general structure Rx-CN- NRy corresponding to the conjugate base of an amine bonded to the N-terminus of a nitrile. Accordingly, Rx and Ry when used in this context may independently be an optionally substituted organic moiety comprising 1 to 12 carbons or any number of carbon atoms falling within this range. Such an organic moiety may comprise the optional substituents as defined for Rj to R ₁₀ .

In an embodiment of the method, there may be a minimal concentration of the probe needed to detect acryloyl groups. This concentration may depend on the amount of the acryloyl groups present in the sample. The concentration of the probe in the sample may need to be at least 1 nM, 10 μΜ or any concentration ranges between 1 nM to 10 μΜ. The concentration of the probe may depend on the concentration of the acryloyl groups that is to be detected. Thus, the concentration of the probe needed for detection may be less than or more than 10 μΜ. The acryloyl groups may need to be present at a concentration of at least 100 nM, 200 nM, 300 nM, 400 nM or 500 nM before introducing the probe to the sample. The minimal concentration of acryloyl groups needed for detection may be at least 100 nM, 200 nM, 300 nM, 400 nM or 500 nM. Meanwhile, the minimal concentration needed for acrylamide to be detected may be 100 nM to 1 μΜ. The minimal concentration for acrylamide to be detected may be lower or higher than the range of 100 nM to 1 μΜ. Accordingly, these concentration limits may differ when it comes to detecting other acryloyl group derivatives. As for the concentration of diaryltetrazole compound to be used, it may need to be at least 1 nM, 10 μΜ or any concentration falling between 1 nM to 10 μΜ. The concentration of diaryltetrazole compound to be used may depend on the concentration of the acryloyl groups. Thus, the concentration of the diaryltetrazole compound needed for detection may be less than 10 μΜ. The concentration of acryloyl groups that needs to be available before they can be detected may also depend on the amount of the diaryltetrazole compounds used.

The probe comprising the diaryltetrazole compound may emit fluorescence when the diaryltetrazole reacts with the acryloyl groups. The intensity of the fluorescence emitted and speed of detection may depend on the factors as discussed above.

The present method may be conducted over the entire pH range i.e. 1 to 14. The present method may be conducted under acidic, neutral or alkaline conditions. Acidic conditions may occur in the range of pH 1 to 6 while alkaline condition may occur in the range of pH 8 to 14. Neutral conditions may occur at pH 7. Accordingly, any one of steps (1) and (2) may be conducted in any of the pH conditions as described above. In a preferred embodiment, the ideal pH range of the sample may be between 8 and 11, since the generation of acrylamide in food matrices has been reported in buffers at high pH. Particularly, the samples may be prepared under alkaline conditions and at specific pH values, such as 8, 9, 10 or 11.

The method may further comprise analyzing pixel values of the fluorescence in the image to obtain a quantification of the acryloyl group in the sample.

The present application further comprises a non-transitory computer readable medium having stored thereon executable instructions for a processor to control the processor perform steps comprising: (1) communicating instructions to a processor of a first device coupled to a second device comprising the processor for detection of the acryloyl group in a sample to performs steps comprising: (a) switching on a first light source of the first device to illuminate the sample and a probe in a compartment of the first device for a first period of time to activate the probe into a reactive intermediate for reacting with the acryloyl group to produce a pyrazoline cycloadduct; and (b) switching on a second light source of the first device for a second period of time to illuminate the pyrazoline cycloadduct so as to excite fluorescence in the pyrazoline cycloadduct, and (2) activating a camera in the second device and coupled to the processor to capture an image of the sample to determine the presence or absence of the acryloyl group in the sample.

The first device for detection of the acryloyl group in a sample that receives instructions from the computer can be the device as described herein.

The second device can be a mobile device having a camera as mentioned earlier in the present application. The non-transitory computer readable medium can be stored in the second device to control the processor of the second device to communicate instructions with the processor of the first device, i.e. the device for detecting the presence or absence of an acryloyl group in a sample, to automate execution of one or more steps of the present method.

Examples

Non-limiting examples of the invention will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention. All chemicals and solvents were purchased from commercial sources including Sigma- Aldrich Corp. (St. Louis, Missouri, United States of America), Alfa Aesar (Haverhill, Massachusetts, United States of America), Fluka (Milwaukee, Wisconsin, United States of America) and JT Baker (Phillipsburg, New Jersey, United States of America) and used directly without purification. 1H NMR spectra were recorded with Bruker Avance III 400, and chemical shifts were reported in ppm using either TMS or deuterated solvents as internal standards (TMS, 0.00; CDC13, 7.26; C6D6, 7.15; DMSO-d6, 2.50). Multiplicity was reported as the follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, b = broad. 13C NMR spectra were recorded at 75.4 MHz, and chemical shifts were reported in ppm using the deuterated solvents as internal standards (CDC13, 77.0; DMSO-d6, 39.5; C6D6, 128.0). LC-MS analysis was performed using Waters 3100 Single Quadrupole LCMS System. Fluorescence detection was performed with Tecan Infinite M1000 and the custom mobile detection platform described below. Food products were purchased from local supermarkets and fast food restaurants. UV-B laser diode (E _max = 310nm) was purchased from Zhuhai Tianhui Electronic Co., Ltd (Zhuhai, Guangdong, China). UV-A light-emitting diode was purchased from Radioshack (Texas, United States). A filter (Lee filter 088) was purchased from PNTA (Seattle, Washington). A Corning 96-well plate (black) was purchased from Sigma-Aldrich Corp. (St. Louis, Missouri, United States of America). The fluorescence microplate readers were purchased from Tecan Group Ltd (Mannedorf, Switzerland).

Example 1 - Synthesis and Characterization of Probe

Scheme 1 below is used to form a fluorogenic probe 1 - diaryltetrazoles from a solution mixture of 4-formylbenzoic acid in ethanol and benzenesulfonohydrazine.

Scheme 1

4-formylbenzoic acid (1.000 g, 6 mmol) was dissolved in ethanol (100 mL), followed by addition of benzenesulfonohydrazine (1.160 g, 6 mmol). The mixture was stirred at room temperature for 1 hour, then quenched with water (100 mL) and stirred for 15 minutes at room temperature. Precipitate was filtered, washed with cold ethanol and dissolved in pyridine (30 mL) to form solution A. Aniline was then dissolved (0.587 g, 0.60 mL, 5 mmol) in water: ethanol (1 : 1, 8 mL) and concentrated HC1 (1.3 mL). NaN02 was dissolved (0.455 g) in water (2 mL). Both mixtures were cooled in ice bath for 5 minutes before addition of NaN02 solution to aniline solution drop wise in an ice bath to form solution B. Solution B was added to solution A drop wise in an ice bath. Mixture was then stirred for 1 hour at room temperature. Mixture was then extracted three times with ethyl acetate (100 mL). 3 M HC1 (250 mL) was added to the combined organic layers and stirred vigorously for 10 minutes. Solvent was removed and then dissolved in dichloromethane. Product was precipitated with hexanes. Product was further washed with cold hexanes to obtain a red solid of diaryltetrazole (0.820 g, 45%). 1H NMR (400 MHz, methanol-d4) δ 8.39 - 8.34 (m, 2H), 8.27 - 8.22 (m, 4H), 7.71 - 7.66 (m, 2H), 7.63 (d, J = 7.3 Hz, 1H). 13C NMR (101 MHz, MeOD) δ 147.01, 141.50, 141.48, 129.93, 129.60, 127.95, 127.89, 127.44, 127.41, 125.52, 119.62. HRMS (ESI) calculated for C14H10N4O2 266.0805 [M + H+], found 266.0804.

Example 2 - Detection of Acrylamide Using Fluorogenic Probe 1

Each sample was reacted with 100 μΜ of the probe 1 as prepared above, in a 25 mM pH 8 sodium phosphate buffer under a UV lamp (302 nm, 1.6 W output) for 1 minute. The sample is then loaded onto a black 96-well plate and analyzed using a fluorescence microplate reader. Standard curves are plotted using freshly prepared acrylamide solutions for each experiment. The standard curves can be subsequently used by the device and method for quantification of the detected acryloyl group.

Example 3.1 - Detection of Acrylamide Using Fluorescence Assay With A Plate Reader In Accordance With An Embodiment of the Present Application

A fluorescence assay based on a photoactivated chemical probe is utilized and this probe specifically reacts with the alkene group found in acrylamide, upon which a highly fluorescent product is formed. The chemical probe is the diaryltetrazole-based probe 1, as discussed earlier, that is non-fluorescent and stable at room temperature. As a result, the background of the reaction is inherently low. Upon a one-minute photo-irradiation using a UV-B source (e.g. a 302 nm lamp), the probe 1 undergoes a cycloreversion reaction, resulting in a reactive nitrile imine intermediate. The reactive intermediate of the probe 1 reacts with acrylamide to form a pyrazoline cycloadduct 2. The reaction scheme between the probe 1 and acrylamide.is shown in Fig. 1A.

The pyrazoline product, i.e. pyrazoline cycloadduct 2, is highly fluorescent, with an excitation maximum at a wavelength of 364 nm and emission maximum at a wavelength of 511 nm. Formation of the fluorescence product in the presence of probe 1 and acrylamide is shown in Fig. IB. As shown, sample 1 comprises only acrylamide; sample 2 comprises only the probe 1 ; sample 3 comprises the probe 1 and acrylamide without UV activation; and sample 4 comprises the probe 1 and acrylamide after UV activation. All samples are placed under a handheld UV lamp to induce fluorescence emission in the pyrazoline product. The results in Fig. IB show that only the sample 4, where the probe 1 and acrylamide have been presented under UV activation before being placed under the UV lamp for UV excitation, can produce the fluorescent pyrazoline product.

Fluorescence characterization of acrylamide detection with the probe 1 is shown in Figs. 1C and ID. The spectral properties of the pyrazoline product are characterized on a fluorescence plate reader. The pyrazoline product is obtained from reaction of 100 μΜ probe 1 and 100 μΜ acrylamide. As shown in Fig. 1C, the molecule of the pyrazoline product 2 is found to absorb strongly in the UV, with an excitation maximum at a wavelength of 364 nm (see Fig. 1C) after reaction with acrylamide. Although an ultraviolet A (UVA) light-emitting diode (LED) used in the device for detection of acryloyl group (will be described below) has an emission maximum at a wavelength of 405 nm, its spectrum is wide enough that there is adequate overlap with the absorbance. Likewise, Fig. ID shows that the emission maximum of the pyrazoline product is at a wavelength of 511 nm. Consequently, all fluorescent readings from the plate reader were recorded at 511 nm with a bandwidth of 5 nm.

As shown in a quantification of acrylamide in Figs. 2 A and 2B, the probe 1 is very sensitive with a lower detection limit of 100 nM or 7 ppb acrylamide in the final solution. Images of acrylamide samples reacted with Probe 1 after photo activation, at concentrations of 100 nM to 1 μΜ (100 nM increments, see the top of Fig. 2A) and 1 to 10 μΜ (1 μΜ increments, see the bottom of Fig. 2A), are shown in Fig. 2A. As shown in Fig. 2A, the same samples are analyzed using a fluorescence plate reader. The resulting fluorescence is highly linear through at least two orders of magnitude. The results in Figs. 2A and 2B show that the present fluorescence assay using the probe 1 in the present application delivers comparable detection ability to the existing state-of-the-art methods like GC-MS/MS or LC-MS/MS. Moreover, the reaction of the probe 1 with acrylamide is quick and can be completed in 1 minute using a UVB lamp. The reaction can also be performed easily in aqueous conditions and is amenable to high throughput screening format using multi-well plates and fluorescence microplate readers.

Example 3.2 - Detection of an Acryloyl Group Using Fluorescence Assay With A Plate Reader In Accordance With Another Embodiment of the Present Application

Since the fluorescence method is able to detect acrylic acid and acrylamide, its versatility in detecting molecules of similar structure is also of interest. The probe is indeed able to detect the presence of molecules such as methacrylamide, methyl acrylate and maleic acid monoamide, thereby suggesting its use in monitoring their concentrations, for both industrial and environmental purposes.

Fig. 2C shows a detection of other acrylates with an acryloyl group (100 μΜ probe 1 + 100 μΜ acrylates) using the probe 1 (see right bars 202, 204, 206, 208, 210, 212 of Fig. 2C). In the absence of the probe 1, or the other acrylates with an acryloyl group, no fluorescence is detected (see left bars 201, 203, 205, 207, 219, 211 of Fig. 2C). The versatility of the present probe 1 is tested using a variety of different acrylates. All samples were dissolved in IX phosphate buffered saline (PBS), to a final concentration of 100 μΜ for both probe and acrylate, and activated for 1 minute as described above. The resulting fluorescence at a wavelength of 511 nm is detected using the fluorescence plate reader, with an excitation wavelength of 364 nm. All the acrylates in Fig. 2C show significant fluorescence, indicating the ability of the probe 1 to react with other targets. It should be noted, however, that these species are not typically found in food products, and thus, will not affect the present method adversely. The experiment results in Fig. 2C also suggest alternative applications for this probe in, for example, industrial processes.

Furthermore, it is appreciable to those skilled in the art that it is the acryloyl group in these acrylates that reacts with a reactive intermediate of the probe 1 to produce a fluorescence product (e.g. the pyrazoline cycloadduct). It is also worth noting that these molecules with the acryloyl group are not typically found in food products, and are thus unlikely to result in spurious signal using the present method. Example 4.1 - Detection of An Acryloyl Group Using Fluorescence Assay With A Device for Detection of An Acryloyl Group In Accordance With A Further Embodiment of the Present Application

The ease of use of the probe 1 in an acryloyl group detection lends itself to a more distributed model of monitoring. For example, by virtue of a device for detection of an acryloyl group as designed in accordance with a further embodiment of the present application, the detection now, instead of being conducted in a centralized laboratory, can be advantageously deployed on-site, for example in food manufacturing facilities to provide rapid feedback when optimizing one's processes (baking times, temperatures, etc.), so as to minimize acrylamide generation.

To demonstrate the feasibility of this embodiment, a device for detection of an acryloyl group has been developed. Detailed design of the device for detection of an acryloyl group is depicted in Figs. 3A, 3B, 3C, 3D and 3E.

In an example shown in Fig. 3A, the device for detection of an acryloyl group can be an attachment device 300 that is able to attach with a mobile phone to realise the detection. In this example, the attachment device 300 comprises two parts- an upper layer 320 and a bottom layer 322- each of which has a footprint barely larger than the mobile phone (e.g. an iPhone 5 in this example). The two parts can be 3D-printed or otherwise manufactured.

The upper layer 320 contains optical components such as a macro lens 318 and an optical filter 316 for detection, and serves as the interfacing layer 320 to interface with the mobile phone. The optical filter 316 is a gel filter. The macro lens 318 can drastically reduce the minimum focal distance of the camera of the mobile phone, permitting a much sleeker profile for the attachment device 300. A sample having an acryloyl group can sit in a compartment 302 of the device 300 just 5 mm from the camera, and the gel filter 316 is selected to attenuate the UV excitation source 306, while allowing the green fluorescence to pass through.

The bottom layer 322 houses electronics, which communicates the mobile phone, and also the compartment/sample chamber 302 for receiving a container comprising the probe 1 with the sample. The compartment/sample chamber 302 can be modified for different assay formats. That is, the compartment/sample chamber 302 can be configured to comprises various types of container, such as a cuvette, a microcentrifuge tube, or a dipstick. The communication between the electronics of the device 300 and the mobile phone is via a wireless connection, e.g. a Bluetooth connection configured by a processor 308 amongst the electronics.

In this embodiment, the processor 308 is a microcontroller programmed using the Arduino programming language, and has a built-in Bluetooth Low Energy (BLE) interface, through which the mobile device communicates with the device. A camera software application can be provided in the present application to allow the mobile device to be pre-programmed to activate the probe 1 for a fixed period of time (e.g. by turning on the UV-B LED for 15 minutes) before snapping a picture to record the fluorescence upon excitation. One can increase the number of UVB laser diodes to further reduce the time required for activation. Likewise, one can also increase the number of UBA laser diodes to further reduce the time required for excitation. The microcontroller may also have a microUSB interface that can be used for software updates to the device.

As shown in Fig. 3 A, the device 300 can comprise a charging circuit 310 used to power the device 300 off a USB 5V source, while also charging an internal battery 312. The internal battery 312 can be a lithium polymer battery or other similar battery that is applicable in the context of a device with a concise structure and the relevant power requirement. In this example, because the operating voltage of the UVB LED 304 is 9V, a voltage regulator 314 is used to step-up the 3.7 V battery source to power the UVB LED 304. The voltage regulator 314 may also contain a logic input (not shown) to allow the UVB LED 304 to be switched on and off. The UVA LED 306 on the other hand runs directly off the 3.3 V digital output of the processor 308 that is connected to the charging circuit 310. The internal lithium polymer battery 312 can power the device 300 for more than 1 day on standby, and at least 3 hours of continuous operation with both LEDs 304, 306 turned on.

In this example, specifications of the electronics used in the device are listed in Table 1 below.

Table 1. List of parts used in the embodiment of the device 300 as shown in Fig. 3A

When in use, the device 300 is attached to a mobile device, e.g. an Apple iPhone 5, as shown in Fig. 3B. The mobile device has a transceiver (not shown) that communicates instructions with the processor 308 of the device 300 via a wireless Bluetooth connection. It can be appreciated by the skilled person that the transceiver of the mobile device 300 can be in other types of communication connection with the processor 308 or other electronic components of the device 300.

As shown in Fig. 3B, the device 300 comprises a housing/enclosure that can encompass a portion of the mobile device (e.g. Apple iPhone 5) as well as the compartment 302, the UVB LED 304, the UVA LED 306 and the electronic components of device 300 for detection of an acryloyl group. The mobile phone, in this case an Apple iPhone 5, is positioned on top of the enclosure, and connects wirelessly to the electronics within.

A simple software application that controls the two LEDs 304, 306, and which allows full manual exposure controls has also been developed to acquire and process the fluorescence signal, yielding a concentration estimate for each sample. The software application can be installed on the mobile device to send instructions to the device 300 to control the two LEDs 304, 306 for the exposure and activate the camera of the mobile device for the acquiring and processing of the fluorescence signal.

The device 300 may include a removable lid 324 through which a container such as a cuvette comprising the probe 1 with a sample can be slid into the compartment 302 for photoactivation and detection, as shown in Fig. 3C.

When the device 300 is coupled to the mobile device (e.g. Apple iPhone 5), a cross- sectional view of the device 300 is shown in Fig. 3D. The cross-sectional view shows the UVB LED 306 for photoactivation, the UVA LED 306 for photoexcitation, and the optical filter 316. In this embodiment shown in Fig. 3D, the optical filter 316 is a green emission. The macro lens 318 is also shown in the cross-sectional view help focus the sample to be imaged by a camera of the Apple iPhone 5. The trapezoid shape indicated by the dashed lines in Fig. 3D refers to the field-of-view of the camera of the mobile device. As described above, it is readily understandable to the skilled person in the art that the apart from Apple iPhone 5, other mobile devices can also be used to work in conjunction with the device 300 for detection. As described earlier in the present application, the mobile device may be other smartphones or tablets with a camera and a mobile operating system, such as Windows of Microsoft, iOS of Apple Inc. or Android of Google Inc.

Example 4.2 - Activation and Detection of Acrylamide using the Device for Detection of An Acryloyl Group as described in Example 4.1

As described above, a UVB LED 304 (Emax = 310nm) is incorporated into the device 300 for detection of an acryloyl group to perform the activation of the probe. Since the power output (1 mW) of the UVB LED 304 is much lower than that of a UV lamp (1.6 W), the ability of the LED 304 to activate the probe in a reasonable time is tested. To do this, a mixture of the probe 1 and acrylamide is placed in a cuvette, and exposed continuously to the UVB source. Fluorescence readings are taken at 5 -minute intervals by turning off the UV-B source, and turning on the UVA LED 306 (Emax = 405 nm). The fluorescence is compared to another sample that has been activated using the UV lamp for one minute in accordance with Example 2.

As described above, each activated sample is illuminated using the UVA LED and imaged with the camera of the mobile device through a macro lens 318 and a filter 316. A software application (e.g. a mobile app) is developed to control the LEDs 304, 306and capture the images. Manual exposure settings can be used to ensure reproducibility of the results (ISO = 736, exposure duration = 0.4 s, colour temperature = 5500K, tint = 0). Since the response curve of the camera to incident fluorescence is non-linear, a linearization procedure is performed.

Despite the far lower output power of the UVB LED compared to the conventional UV lamp (around 1600 times lower), in the present example, the activation of the probe is completed within 15 minutes (see Figs. 4A-B) using the device 300. This is because the total area of the UV lamp tube is around 130 cm2, but that of the sample is only around 1 cm2. As a result, only 1% of the UV lamp irradiation is absorbed by the sample. Therefore, a much lower power LED 304 can be advantageously used by running off a small lithium polymer battery 312 to perform the same task in a reasonable amount of time. One can also increase the number of laser diodes to further reduce the time required for activation.

The fluorescence detection of the sample is carried out using a built-in 390-410 nm UV-A LED as an excitation source, and using filtered camera of the mobile device as the detector. Similar to UVB LED, one can also increase the number of UVA laser diodes to further reduce the time required for activation. In the present example, to capture the image, the sensor sensitivity (ISO) of the camera of the mobile phone can be set to the maximum value (736) while the exposure duration is set to 0.4 s to yield the appropriate dynamic range. Since the camera of the mobile device is not designed for scientific applications, the response curve is non-linear. However, linearization of the raw green pixel value G _raw can be achieved by the following equation:

G _lin = 255 * (G _raw /255) ^2'2

Plotting the G _un values against the concentration of acrylamide yields a good linear fit (R ² > 0.99), and is sufficient for quantification of the acrylamide content. Fig. 4C shows that the resulting fluorescence is highly consistent among separate preparations, as evidenced by the small variation in the detected signal (coefficient of variation, CV < 10%).

This in turn advantageously allows users to determine an absolute concentration of the acrylamide in the sample without also needing to prepare a standard curve with every new sample. However, since the curve has a positive x-intercept as shown in Fig. 4C, the limit of detection on the device is around 0.7 μΜ in the final sample solution, corresponding to about 0.1 ppm for raw sugar, and 0.5 ppm for other food products according to an extraction protocol of the present application.

Example 6 - Sample Preparation for Food Samples

Food samples (potato chips, cereal, French fries and raw sugar) were prepared using the QuEChERS method with some modification. Briefly, 5 mL of hexane is added to 1 g of potato chips and cereal (finely crushed with mortar and pestle) or French fries (cut to small pieces) in a 50 mL centrifuge tube and vortexed for 1 minute. Water and acetonitrile (10 mL each) are then added, followed by the Bond Elut QuEChERS salt packet (Agilent Technologies). After shaking the mixture vigorously for 1 minute, the tube is placed in a centrifuge and spun down at 5,000 rpm for 5 minutes. The top hexane layer is then removed to de-fat the sample. 1 mL of the acetonitrile layer is removed and placed in a 2 mL microcentrifuge tube containing the Bond Elut AO AC dispersive solid phase extraction (dSPE) sorbent (Agilent Technologies), and vortexed for 1 minute (Fig. 5). The sample is then spun down for 1 minute at 5,000 rpm, whereupon 0.5 mL is removed and mixed with 0.5 mL of water. Finally, this mixture is evaporated in a SpeedVac (Eppendorf) to remove the acetonitrile, and the volume is adjusted back to 0.5 mL to complete the sample extraction and solvent exchange into water. This sample is then reacted with our probe (final concentration = 100 μΜ) in 25 mM pH 8 phosphate buffer, and analyzed on a plate reader (volume = 100 μί) and the mobile platform. Each sample is tested in triplicate. Raw potatoes were sliced and lyophilized, and the matrix crushed to serve as negative controls for the chips. Because of its high solubility in water, a larger quantity of raw sugar is added to improve the detection limit (4.5 g sugar in 10 mL water). Omission of the hexane extraction step for raw sugar did not significantly affect the extraction results. To determine the overall extraction efficiency of the procedure, 10 μΜ of acrylamide added directly to the food matrix, air-dried for 30 minutes and processed as described above. The difference between the spiked-in samples and those without any added acrylamide can then be used to determine the extraction efficiency. This can in turn be used as a correction factor in the calculation of the acrylamide content in each food sample. Similarly, acrylamide is added at various stages (before dSPE, and after dSPE extraction) to verify the acrylamide recovery rate of each step.

Each food sample was also sent to a commercial testing laboratory for acrylamide content quantification (ALS Technichem (S) Pte Ltd, QWI FD/FC93 by LC-MS/MS characterization). An additional potato chip sample with 10 ppm of acrylamide spiked in was also sent to determine the acrylamide extraction efficiency for their method.

Example 7 - Detection of Acrylamide in Food Samples

As a highly water-soluble small molecule, extraction of acrylamide from pulverized food matrices by simple immersion in aqueous buffers is highly efficient. However, the complex composition of these matrices means that many other species are co-extracted with acrylamide. Although the present detection can be reliably performed in most aqueous solvents, and can tolerate a high fat content, these co-extractives result in unacceptably high background which can obscure the signal from the assay (λ Em= 510 nm).

Figs. 4D and 4E shows a comparison between the aqueous and QuEChERS extraction protocols on a potato chip sample. A simple aqueous extraction method and the more involved QuEChERS approach were employed to extract the acrylamide from the food matrices. For the aqueous extraction method, crushed chip or cereal sample (~ 0.5 g) was loaded into a 5 mL syringe, and 2 mL of pH 8 sodium phosphate buffer was drawn into the syringe. The samples are then shaken briefly by hand, ensuring that the food matrices are fully submerged in the buffer, and left to stand for 20 minutes. The mixture is then passed through a 0.22-micron syringe filter, and the resulting extract is tested for acrylamide. Each sample is diluted 10-fold in additional pH 8 buffer, and reacted with 100 μΜ probe 1 under UV-B activation. The resulting fluorescence is then tested on the device of the present invention and corroborated with readings from a microplate reader. To determine the extraction efficiency of this approach, 100 nmoles of acrylamide (corresponding to an additional 5 μΜ in final concentration) is spiked into chips and cereal samples and compared with samples without spike-in. To verify that the complex food extract does not interfere with our reaction, comparison with post-extraction spike-in of 5 μΜ acrylamide is also performed.

The aqueous method is very straightforward, and it is verified that the acrylamide that was spiked-in post-extraction is detected with our probe (4.5-5 μΜ, or 90-100%), indicating that the final reaction solution does not impede acrylamide detection. In addition, around 50% of acrylamide spiked-in pre- extraction was recovered, a number that is comparable to the 50-60% recovery of the QuEChERS approach (see Fig. 4E).

However, the simple aqueous extraction produces a sample that is strongly coloured (yellowish-brown), suggesting the presence of a large quantity of co-extractants. Indeed, these unidentified species yielded strong fluorescence in the blue-green region after exposure to UV-B, even in the absence of the probe 1 (see Fig. 4D). Addition of the probe 1 yields acrylamide content estimates corresponding to 10-20 ppm, numbers which are too high to be reasonable. For example, for the BBQ chips, the estimated acrylamide content (-15 ppm) is almost 2-3 times as high as the maximum expected for normal food products, and represent values only seen in overcooked oil-fried chips. While this cannot be ruled out, it is far more likely that among the co-extractants are other species that also react with the probe and thus yield fluorescent products. The QuEChERS approach, on the other hand, yields colorless and clear samples, and when exposed to UV-B the sample without probe is essentially non-fluorescent (see Fig. 4E). As discussed earlier in the present application, addition of the probe provides estimates of acrylamide content that are within the reported values, though the extent to which residual co-extractants contribute to the signal has to be studied in greater detail with a better sample extraction protocol that can separate acrylamide from them.

To minimize this extraction artifact, a modified QuEChERS protocol is performed (see Fig. 4F) that results in significant reduction of background fluorescence in the spectral range of interest (see Fig. 4E). The solvent exchange step (Fig. 5, Step 3) is necessary to ensure a more consistent signal across the samples (see Fig. 4F). A 1.5x correction factor was factored in to account for the residual acetonitrile, which enhances the fluorescence signal. As shown in Fig. 4F, in the modified QuEChERS protocol, the presence of acetonitrile is found to greatly enhance the fluorescence of the pyrazoline product. However, as acetonitrile evaporates rapidly from the mixture, it can result in large variations in the signal as the acetonitrile concentration changes. Consequently, a solvent exchange from acetonitrile to water is performed in our method. It is worth noting that this enhancement is reversible, and does not appear to be due to any reaction between acetonitrile and the probe, but rather results from solvent effects on the fluorophore.

Although a complete solvent exchange from acetonitrile to water is attempted, the control experiments suggest that around 10% of acetonitrile can remain in the final mixture, resulting in a 50% increase in signal (C.V. < 5%). A correction factor is included in the final calculation to account for this difference. Alternatively, one can allow the residual solvent to evaporate from the mixture, which occurs relatively quickly (30-60 min at room temperature and pressure for a 100 μΕ sample on a 96-well plate).

The ideal pH range of the present assay is between 8 and 11, and since de novo generation of acrylamide in food matrices has been reported in buffers at high pH, we have chosen pH 8 for all our sample preparation steps. Furthermore, to ensure that the pH remains stable after introducing the extract, we increased the buffer ionic strength from 10 mM to 25 mM. Much higher buffer concentrations were found to adversely affect the assay performance, and have thus been avoided. Using this extraction protocol, the detection limit of the method on a microplate reader is around 0.03 ppm for raw sugar, and 0.15 ppm for other food products.

Example 7.1 - Verification of Extraction Protocol using Fluorescence Assay

In order to verify the acrylamide recovery rate of the present method, acrylamide of known concentration is introduced into the food samples. It is found that >90% of acrylamide introduced immediately prior to and after the dSPE step were recovered, indicating that neither the dSPE nor the final solvent exchange into water adversely affected the acrylamide concentration in the sample (see Fig. 5). The successful detection of spiked- in acrylamide also confirmed that the final sample condition (pH, buffer concentration, residual solvents, etc) did not inhibit acrylamide detection using our probe. Overall recovery rate of acrylamide is around 50 to 60% for solid food matrices, which is comparable to reported values. For raw sugar, the overall extraction efficiency is in excess of 85%, probably because the acrylamide does not need to be released from an insoluble matrix and is fully solubilized.

Example 7.2 - Quantification of acrylamide in food samples using fluorescence method

Quantification of the acrylamide content was performed on generic corn flakes cereal, raw sugar, two types of potato chips (original and BBQ flavored), French fries, as well as lyophilized raw potato slices as negative control, using both the plate reader and mobile assay platform (Table 2). The results from both fluorescence detection methods were very similar, confirming the accuracy of the mobile platform.

Acrylamide was undetectable for both the lyophilized raw potato and corn flakes. For the raw potato sample, this is probably due to the absence of acrylamide since it has not been exposed to heat and hence the Maillard reaction. On the other hand, the reported acrylamide levels in corn flakes (0.07 ppm) as well as that determined by LC-MS/MS (0.1 ppm) are below the detection limit of our detection protocol. In all other cases the acrylamide estimates lied within the ranges reported in literature. In particular, raw sugar was tested as a proxy for soluble solids, and was found to yield excellent acrylamide recovery, and an increased sensitivity due to the higher mass that can be processed in each sample preparation.

Table 2. Summary of data

Concentration Concentration Commercial Reported from Plate from Mobile LC-MS/MS Concentration Reader (ppm) Device (ppm) (ppm) Range (ppm)

Corn Flakes N.D. N.D. 0.10 0.07

Raw Sugar* 0.12 + 0.05 0.12 + 0.02 0.08 0.03 - 2.7

Fries 0.60 ± 0.06 0.53 + 0.09 0.37 0.1 - 1.5

Original

Potato 3.4 + 0.19 3.8 + 0.86 2.73 0.35 - 5.0

Chips

BBQ

Flavored 2.2 + 0.32 2.5 + 0.85 1.91 0.35 - 5.0

Potato Chips

Lyophilized

N.D. N.D. 0.04* N.D.

Potato

* The detection limit for raw sugar is higher than that for other samples due to its high solubility in water, allowing a larger mass to be used in each extraction.

* This value is just above the limit of detection of the method, and may be an artifact.

Plotting the acrylamide content estimates using our method against those obtained from LC-MS/MS testing shows that the estimates are reasonably close, but ours were consistently higher. Moreover, the results from the two methods appear to be correlated, suggesting the presence of a systematic error.

Since the two methods are very different approaches to quantifying acrylamide, it is possible that the various artifacts in each method contributed to this error. If the differences are indeed artifactual in nature, a simple linear transformation will be sufficient to correct for them. Testing a larger number of samples using both methods can help refine this linear relationship, though the cost of LC-MS/MS (USD$200 per sample) remains prohibitive.

On the other hand, the differences may be due to the presence of other species that can react with the probe. One possible candidate is acrolein, which a known byproduct of the frying process and which is, owing to its harmfulness, also of concern due to possible health effects. A more thorough investigation and improved extraction protocol will be needed to determine the presence of these species and their contribution to the signal definitively.

As shown in Fig. 6, instead of testing the extracted sample, various samples are sent in their solid forms to a commercial testing laboratory. This allows to compare the current entire workflow (including the device for detection of an acryloyl group) against the commercial LC-MS/MS protocol, which is also employed in nearly every survey of acrylamide content in food. This protocol, as provided by the vendor, is as follows.

A deuterated internal standard, [ ¹³ C ₃ ]-acrylamide or d ₃ -acrylamide, is added to the test portion, followed by extraction of acrylamide with water, protein precipitation with Carrez I and II solutions. The non-polar interference product is eliminated with liquid-liquid extraction (water-dichloromethane). Acrylamide is then extracted with ethyl acetate, which is subsequently passed through a SPE multimode cartridge and a strong cation exchanger. The final quantification is done by LC-MS/MS in multiple-reaction monitoring (MRM) with positive electrospray ionization. Using a sample with 10 ppm acrylamide spiked in, we determined the extraction efficiency of the LC-MS/MS method to be around 56%. This number is used as a correction factor for all other samples.

A comparison of the acrylamide content between the two methods shows that the estimates are reasonably similar. However, there appears to be a systematic error between the estimates, which likely arises from artifacts between the methods that have not been corrected for (Fig. 6).

One possibility is that the residual acetonitrile in the samples is higher than 10%, which results in under-correction of the enhancement and an erroneously high estimate using the fluorescence method. The volatility of acetonitrile may also result in some degree of concentration of the sample after the dSPE step, further exacerbating the problem. Both of these can be eliminated with a more optimized processing protocol.

Example 7.2.1 - Paper detection of acrylamide

One possible strategy is to use chromatography to separate the sample mixture. Preliminary experiments with the probe applied on a cellulose-based filter paper were performed. Upon incubation with an aqueous solution containing acrylamide and UV photoactivation, the fluorescence product can be clearly observed (see Fig. 7). By coupling this to an appropriate solvent system, it may be possible to achieve acrylamide detection even in complex mixtures.

In an example, paper chromatography is employed to separate acrylamide from co- extractants, which may react with the probe. An optimized solvent system and paper treatment remains to be developed and is not further discussed in the present application. However, it is verified that the ability to perform the reaction in a paper format by spotting 1 μL of 100 mM probe on filter paper (labeled ZY in Fig. 7), and air-drying the spots. 1 μL· of 100 mM acrylamide (Aery) and acrylic acid (A A) in different buffers are then spotted on the probe spots and activated under the UV-B lamp for 1 minute (Fig. 7, top). The samples with acrylamide and acrylic acid were much brighter than that with only the probe (Fig. 7, top right sample in each image). A 50X diluted sample was also tested, and found to be only slightly brighter than the probe-only control (Fig. 7, top left sample in each image). The dilution step was necessitated by the high concentration of organic compounds and also pigments in the sample, which obscures the signal. However, the dilution also drastically reduces the signal. An optimized chromatographic separation protocol would address these issues.

Advantages

In addition to the advantages described earlier in the present application, it is worth noting that the total cost of using the present device and method for detection of an acryloyl group would be less than USD$75 in hardware and USD$0.003 per assay, representing a 50,000- fold decrease in cost over existing methods. The device, when attached with a mobile device (e.g. a mobile phone), provides a portable and inexpensive platform for detection of an acryloyl group, especially acrylamide detection. This will advantageously benefit three user groups. First, regulatory and food inspection agencies can simplify their workflow and improve their throughput, while lowering the cost of inspection. While there is currently no regulation that mandates acrylamide testing, it is a service that is provided by many regulatory agencies and testing companies. Second, food companies need to balance flavor through the browning reaction (Maillard reaction) while minimizing acrylamide production. A rapid test to assess food-processing methods onsite allows them to quickly tweak their processes without having to send samples to an analytical laboratory, thereby reducing costs. This will be particularly important to small and medium sized companies for whom the same food safety regulations apply but whose resources may be limited. Third, this will appeal to an increasingly health-conscious consumer market. The portability of the device, as well as the ready-availability of the components, has the potential to democratize monitoring of acrylamide, as well as other contaminants, empowering consumers to make informed decisions on their food choices. To that end, the capabilities of the system can be easily extended and/or modified, e.g. for other fluorescence assays, by distributing software updates via the existing mobile application stores.

In a nutshell, the present application provides a portable sensor device coupled with fluorescence assay to address the needs of an increasingly educated consumer market looking for more transparency in the safety of the products they purchase. Food security will be thereby improved through lowering the cost and complexity of food safety monitoring. While the more immediate users of these technologies may be the regulatory agencies and commercial food companies, the low-cost and convenient device will ultimately empower consumers to make more informed dietary choices.

It will be apparent that various other modifications and adaptations of the invention will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims.