GB2570157A - Molecule detection - Google Patents

Abstract

A method of detecting molecules of a target type in a sample comprises providing an aptamer-nucleic acid duplex, contacting the sample with the aptamer-nucleic acidduplex, wherein the aptamer is capable of selectively dissociating from the nucleic acid to selectively bind to a molecule of the target type in the sample, amplifying any dissociated nucleic acid and detecting any amplified nucleic acid. The method further comprises using the detected result to indicate the presence of molecules of the target type and/or quantify an amount of molecules of the target type. Also provided is a system for detecting molecules of a target type in a sample, wherein the detector is a chemical field-effect transistor (ChemFET) preferably an ion-sensitive field-effect transistor (ISFET) and proton release during amplification is detected. The target may be a peptide such as a hormone, including leptin, ghrelin, CCK, PYY or GLP-1.

Description

(57) A method of detecting molecules of a target type in a sample comprises providing an aptamer-nucleic acid duplex, contacting the sample with the aptamer-nucleic acidduplex, wherein the aptamer is capable of selectively dissociating from the nucleic acid to selectively bind to a molecule of the target type in the sample, amplifying any dissociated nucleic acid and detecting any amplified nucleic acid. The method further comprises using the detected result to indicate the presence of molecules of the target type and/or quantify an amount of molecules of the target type. Also provided is a system for detecting molecules of a target type in a sample, wherein the detector is a chemical field-effect transistor (ChemFET) preferably an ion-sensitive field-effect transistor (ISFET) and proton release during amplification is detected. The target may be a peptide such as a hormone, including leptin, ghrelin, CCK, PYYorGLP-1.

At least one drawing originally filed was informal and the print reproduced here is taken from a later filed formal copy.

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Molecule Detection

Technical Field

The present invention relates to the detection of molecules of a target type in a sample. The present invention also provides a method of identifying a particular condition or status of a person or animal and a method of facilitating the determination of a health or lifestyle change of a subject as well as a system for detecting molecules of a target type in a sample.

Background

The detection and analysis of molecules in a sample is important in the fields of medical diagnostics, forensics and research, amongst others. More recently, such detection has been employed for informing lifestyle choices including choices relating to diet, exercise, and skincare. The presence of a molecule in a sample may be detected by exposing the sample to a ligand and detecting the binding of the ligand to the molecule, if present.

Traditional protein detection techniques include enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays. In an ELISA, the molecule is immobilised by direct adsorption to the assay surface, or alternatively by being captured by an antibody present on the assay surface (sandwich assay). An enzyme conjugated primary antibody can then bind to the molecule, the enzyme enabling direct detection. Alternatively, an unlabelled primary antibody can bind to the molecule. Following this, a conjugated secondary antibody can bind to the unlabelled primary antibody, the conjugate enabling detection. In radioimmunoassays, a known quantity of the molecule is radiolabelled and then mixed with a known amount of antibody specific for the molecule, leading to the binding of the antibody radiolabelled molecule. A sample containing an unknown quantity of the molecule is then added to the mixture, causing the unlabelled molecule from the sample to compete with the radiolabelled molecule for antibody binding sites, displacing the radiolabelled molecule. The quantity of unlabelled molecule is then determined by measuring the radioactivity of the free molecule. These methods are expensive and time consuming, requiring numerous steps, incubation periods, and reagents.

Many protein detection techniques rely on antibodies as the ligand which binds to the molecule, enabling subsequent detection. However, antibodies are expensive to develop and require refrigeration or freezing for storage. In addition, conjugated antibodies require dark conditions when stored.

More recently, aptamers have been considered as ligands for molecule detection. An aptamer is a single stranded molecule that has a specific affinity for a particular target molecule.

Although substantial progress has been made in the field of molecule detection in recent years, there are still unmet needs. For example, there remains a need for a highly sensitive assay which is low-cost, convenient to use and portable.

Summary

According to a first aspect of the present invention, there is provided a method for the detection of molecules of a target type in a sample, the method comprising:

providing an aptamer-nucleic acid duplex;

contacting the sample with the aptamer-nucleic acid duplex, wherein the aptamer is capable of selectively dissociating from the nucleic acid to selectively bind to a molecule of the target type in the sample;

amplifying any dissociated nucleic acid; detecting any amplified nucleic acid; and using the detected result to indicate the presence of molecules of the target type and/or quantify an amount of molecules of the target type.

The present invention thus utilizes the detected amplified nucleic acid as being representative of the amount of the molecule of the target type. This advantageously provides a highly sensitive yet simple assay.

Although suitable for the detection of any molecule of a target type, it is envisaged that this invention will primarily be of use in the detection of molecules of a target type associated with lifestyle, for example, obesity, such as leptin, or fatigue related molecules. The present inventors envisage that the results of such a method can assist a subject in making more informed and healthy lifestyle choices.

In the context of the present invention, “molecules of a target type” will be understood to refer to a plurality of molecules of the same type; i.e. the each of the plurality of the molecules is the same or is considered equivalent.

In some embodiments, molecules of a target type comprise molecules of a biomarker, i.e. each of the plurality of molecules is the same biomarker. As the skilled person will appreciate, a biomarker is a molecule which is associated with a biological process. The process may be that of a normal, healthy biological process, or may be a pathogenic or disease biological process. Hence, detecting the amount of amplified dissociated nucleic acid may be used to determine the amount of molecules of a particular biomarker in a sample. When a sample is obtained from a subject, this may be used to diagnose a patient with a particular disease. Alternatively, the quantification of the amount of molecules of a particular biomarker in a sample may be used to determine whether a pathogen is present in the sample. When the sample is obtained from a subject, this may be used to determine whether or not the subject is infected with the pathogen.

Detecting the amount of molecules of a particular biomarker in a sample may be used as an indicator of the state of a metabolic process, when the sample is obtained from a subject. The state of the metabolic process may be used to determine how healthy the subject is.

The metabolic process state may be associated with the weight, for example obesity, anorexia or bulimia, stress level or fatigue of a subject.

In some embodiments, the target type comprises a small molecule, a nucleic acid, a peptide, a cell or an organism. The organism may be a microorganism, for example a bacterium, a fungus, a virus, an algae, archaea or protozoa. The microorganism may be a bacterium, a fungi or a virus.

A small molecule will be understood to be a biomarker having a low molecular weight. Various small molecules will be known to those skilled in the art, for example, haemin. A low molecular weight may be less than 2000 daltons, less than 1500 daltons, 1000 daltons, less than 900 daltons, less than 800 daltons, or less than 700 daltons.

In some embodiments, the target type comprises a peptide. The target type may comprise a polypeptide. A polypeptide will be understood to be a long peptide chain, for example, a peptide chain comprising more than 50 amino acids. In some embodiments, the target type comprises a protein. As the skilled person will appreciate, a protein comprises one or more polypeptides.

In some embodiments, the target type comprises an enzyme, a hormone, a biological factor, an antigen, a cytokine or a chemokine. A biological factor may comprise a growth factor.

The target type may comprise a hormone. It will be appreciated that some, but not all hormones comprise a peptide or protein. In some embodiments the target type comprises one or more gut hormones. These are hormones comprising protein which are associated with appetite. In some embodiments the target type comprises one or more of leptin, ghrelin, CCK, PYY and GLP-1.

In some embodiments the target type comprises leptin. Leptin is a hormone made by adipose cells, which has been shown to play a role in the regulation of appetite and metabolism. Leptin is known to interact with receptors in the hypothalamus part of the brain and control appetite (Pan HT et al., 2014).

In the context of the present invention, “nucleic acid” will be understood to refer to a sequence of nucleotides. It will be appreciated that the nucleic acid can include any suitable number of nucleotides. The nucleic acid may be double or single-stranded. The nucleic acid may be single-stranded.

The nucleic acid may comprise or consist of DNA, RNA, XNA or combinations thereof. As the skilled person will appreciate, the term “XNA” refers to artificial nucleic acid analogues. Artificial nucleic acid analogues can be distinguished from naturally occurring nucleic acid (such as DNA or RNA), due to changes to the backbone of the nucleic acid in the XNA. Hence, the term “XNA” may refer to a nucleic acid comprising one or more artificial nucleotides. In some embodiments the nucleic acid comprises or consists of DNA or RNA. In some embodiments the nucleic acid comprises or consists of DNA.

In the context of the present invention, the term “aptamer” will be understood to refer to a single-stranded molecule that can selectively bind to a molecule of a particular target type. In some embodiments the aptamer is not naturally occurring. The aptamer may selectively bind to the molecule of a particular target type by a mechanism which is independent of Watson/Crick base pairing or triple helix formation.

The aptamer may be a nucleic acid or a peptide. It will be appreciated that an aptamer can include any suitable number of nucleotides, when a nucleic acid, or any suitable number of amino acids, when a peptide. In some embodiments, the aptamer comprises or consists of a nucleic acid. The nucleic acid may comprise or consist of DNA, RNA or XNA.

In some embodiments the aptamer may comprise or consist of XNA. The provision of at least one artificial nucleotide in an aptamer can be useful for increasing the aptamer’s affinity for a molecule of a target type. The provision of at least one artificial nucleotide in an aptamer can also protect against degradation by nucleases which may be present in the sample.

In some embodiments the aptamer comprises or consists of DNA.

In some embodiments the aptamer comprises or consists of RNA.

In the context of the present invention, “an” or “the aptamer” refers to a plurality of one type of aptamer, each of which having the same or equivalent sequence, each aptamer being able to selectively bind to a molecule of a target type. “Aptamers” refer to more than one different type of aptamer. Different aptamers can have either the same or different numbers of nucleotides or amino acids, and thus can have either the same or different length sequence.

By “aptamer-nucleic acid duplex”, it will be understood that the nucleic acid and the aptamer are bound to each other.

In embodiments where the aptamer comprises a nucleic acid, it will be understood that the nucleic acid of the duplex has a sequence complementary to the nucleic acid sequence of the aptamer. It will thus be understood that because the sequence of each is complementary to the other, the aptamer comprising a nucleic acid and the nucleic acid are bound to each other by Watson-Crick base pairing.

Suitable methods of identifying aptamers will be known to those skilled in the art. For example, nucleic acid aptamers can be identified from a candidate mixture of nucleic acids, where the aptamer is a ligand of the target, by Systematic Evolution of Ligands by Exponential enrichment (SELEX) methods. SELEX methods involve (a) contacting the candidate mixture with the target, wherein nucleic acids having an increased affinity to the target relative to other nucleic acids in the candidate mixture can be partitioned from the remainder of the candidate mixture; (b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and (c) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids, whereby aptamers of the target molecule are identified. Other suitable methods for identifying/selecting aptamers will be known to those skilled in the art.

By “selectively bind” it will be understood that the aptamer has selectivity for a molecule of one target type over other target types (if present) in the sample. It is recognized that affinity interactions are a matter of degree; however, in the context of the present invention, the “selective binding” of an aptamer for its molecule of a target type means that the aptamer binds to its molecule of a target type generally with a much higher degree of affinity than it binds to other, non-target, components in a mixture or sample. In some embodiments, the binding affinity of the aptamer for its respective target type is at least 2X, at least 5X, at least 10X, at least 50X or at least 100X greater than for the other target types (if present) in the test sample. By “selectively dissociating” it will be understood that the aptamer has a greater affinity for the one target type than for the nucleic acid with which it forms the duplex. Hence, in the presence of the one target type in the sample, the aptamer selectively dissociates from the nucleic acid to instead bind to a molecule of the target type. In some embodiments, the binding affinity of an aptamer for a molecule of a target type is at least 2X, at least 5X, at least 10X, at least 50X or at least 100X greater than the binding affinity of the same aptamer to the nucleic acid with which it forms a duplex.

The term “dissociating” will be understood to refer to the breaking of any bonds between the aptamer and the nucleic acid. This is due to the greater affinity the aptamer has for the molecule of the target type as compared to the nucleic acid. The molecule of the target type can therefore be considered to displace the nucleic acid from the aptamer-nucleic acid duplex. Hence, when the aptamer has selectively bound to a molecule of the target type in the sample, this may be understood to be an aptamer-molecule duplex.

Without wishing to be bound by theory, the present inventors believe that all molecules of a target type in the sample will each bind to an aptamer which is capable of selectively binding a molecule of the target type. The present inventors therefore believe that the amount of dissociated nucleic acid and hence the amount of amplified nucleic acid is directly proportional to the amount of molecules of a target type present in the sample.

Amplifying any dissociated nucleic acid will be understood to refer to a method of making multiple copies of dissociated nucleic acid. The amplified nucleic acid may be single or double stranded. In embodiments, the amplified nucleic acid comprises or consists of DNA or RNA. The amplified nucleic acid may comprise or consist of DNA. The amplified nucleic acid may comprise or consist of double stranded DNA.

Methods of amplification are well known to those skilled in the art. Amplifying any dissociated nucleic acid may comprise thermally amplifying any dissociated nucleic acid. In some embodiments thermally amplifying any dissociated nucleic acid comprises thermocycling or isothermal amplification. As will be known by those skilled in the art, “isothermal amplification” refers to the replication of a target nucleic acid sequence wherein all parts of the reaction, including the annealing of the primer and the extension thereof by a polymerase enzyme and, optionally, the denaturation of double-stranded DNA to provide a single-stranded target sequence, are carried out at a single temperature. Thermal amplification methods provide a convenient way of amplifying any dissociated nucleic acid which is low-cost and simple to operate.

An example of thermocycling is the polymerase chain reaction (PCR). For example, in a PCR, first the nucleic acid is optionally exposed to a temperature that is high enough (about 95 °C) to denature the nucleic acid by separating the two strands (if the nucleic acid is double-stranded). The temperature is then reduced (to about 55 °C) to allow annealing (i.e. binding) of primers to the single nucleic acid strands. Then the temperature is increased (to about 72 °C) so that a polymerase enzyme starts synthesizing a copy strand by extension of the primer bound on the template nucleic acid. Ideally, at each stage of the amplification, the number of nucleic acid copies doubles.

Unlike PCR, isothermal amplification techniques operate at a fixed temperature. The temperature is selected to allow hybridization of the designed primer to the nucleic acid template. Isothermal amplification methods include, but are not limited to, loopmediated amplification (LAMP), nucleic acid sequence-based amplification (NASBA), helicase-dependent amplification (HDA), strand displacement amplification (SDA), and rolling circle amplification (RCA).

The skilled person will be aware of suitable primers for each method of amplification. In some embodiments, the primers and/or probes (for example TaqMan probes) will be provided pre-bound to the nucleic acid of the aptamer-nucleic acid duplex. Hence, the association of the primer and/or probe with the nucleic acid can occur prior to contacting the sample with the aptamer-nucleic acid duplex. Conveniently, this results in a fast assay since the initial amplification step of annealing the primer and/or probe to the nucleic acid has already been performed.

Alternatively, the primers may be provided separately to the duplex. For example, the primers and/or probes may be provided in a separate chamber to the duplex. Thus, the primers and/or probes may only anneal to the nucleic acid of the duplex once the nucleic acid has dissociated and has travelled to the chamber in which the primers and/or probes are found.

It will be appreciated by those skilled in the art that methods of amplification including, but not limited to, PCR or LAMP may generate double stranded amplified nucleic acid. For the generation of single stranded amplified nucleic acid, the skilled person will be aware of asymmetrical amplification methods, for example NASBA.

As will also be appreciated by those skilled in the art, each type of thermal amplification method is typically associated with certain enzymes and reagents. Conveniently, therefore, prior to amplifying any dissociated nucleic acid, the method may comprise a step of providing the required enzymes and reagents for amplification.

In some embodiments amplifying any dissociated nucleic acid and detecting any amplified nucleic acid occur at substantially the same time, for example, in a real-time amplification method. This means that it is not necessary to wait until all of the dissociated nucleic acid has been amplified before carrying out detection. Advantageously, this results in a fast assay.

In some embodiments thermally amplifying any dissociated nucleic acid comprises qPCR (quantitative PCR) or l_AMP. qPCR is a PCR wherein the amplification of the nucleic acid is measured in real-time, in contrast to a conventional PCR, where the amount of amplified nucleic acid is measured at the end of the reaction. It will be appreciated that LAMP can also be carried out as a real-time method. Since the amplification is measured in real time, amplifying any dissociated nucleic acid and detecting any amplified nucleic acid can occur at the same time.

LAMP may be carried out at a fixed temperature of approximately 60-65°C.

In some embodiments detecting any amplified nucleic acid further comprises quantifying the amount of amplified nucleic acid. Suitable methods for quantification will be known to those skilled in the art.

In some embodiments detecting any amplified nucleic acid comprises detecting any amplified nucleic acid using a chemical field-effect transistor (ChemFET). ChemFET acts a chemical sensor for the detection of atoms, molecules or ions.

The ChemFET may comprise an ion-sensitive field-effect transistor (ISFET). It will be appreciated that when the ion concentration in a sample to be measured changes, the voltage on the ISFET gate is modulated, which can be detected by the ISFET when suitably biased. One example of an ISFET is the Genalysis® technology by DNA Electronics (DNAe), London, UK.

In some embodiments, detecting any amplified nucleic acid comprises optically detecting any amplified nucleic acid. Optically detecting any amplified nucleic acid may use a complementary metal-oxide semiconductor sensor (CMOS) or semiconductor charge-coupled device (CCD) sensor.

Optically detecting any amplified nucleic acid may comprise providing a label which enables the optical detection of any amplified nucleic acid. Suitable labels include radiolabels, colorimetric labels and fluorescent labels, such as intercalating dyes. For example, the intercalating dye SYBR Green (ThermoFisher Scientific), fluoresces when bound to double stranded DNA. The label may be attached to a probe which is capable of interacting with the dissociated nucleic acid, for example, TaqMan (ThermoFisher Scientific) or molecular beacon based detection. Molecular beacons are available commercially, for example from Merck. TaqMan or molecular beacon based detection may be used to detect amplified single stranded nucleic acid. Techniques such as fluorescence polarization and FRET can be used to detect interaction between a probe and its respective dissociated nucleic acid or amplified nucleic acid

Other techniques can be used to detect interaction between a probe and its respective dissociated nucleic acid or amplified nucleic acid which do not require the probe to comprise a label, for example surface plasmon resonance.

It will be appreciated that, during amplification, every time a single nucleotide is incorporated into the forming amplified nucleic acid, there is a release of pyrophospate ion. It is known in the art that pyrophosphate ion can combine with divalent metallic ion to form an insoluble salt (Tomita et al., 2008). The salt can be optically detected by methods known to the skilled person, for example, by the addition of a fluorescent metal indicator such as manganous ion and calcein. Thus, in some embodiments, optically detecting any amplified nucleic acid comprises detecting a release of pyrophosphate ion, for example by detecting an insoluble salt.

In some embodiments detecting any amplified nucleic acid is label-free, for example, when ChemFET is used. This reduces the cost and complexity of the method.

It will be appreciated that, during amplification, every time a single nucleotide is incorporated into the forming amplified nucleic acid, there is a release of protons. Hence, in some embodiments, detecting any amplified nucleic acid comprises detecting a release of protons from the amplified nucleic acid. This provides a highly sensitive, real-time assay.

Since detecting and/or amplifying the dissociated nucleic acid can be carried out in real-time, this provides a fast yet simple assay which can advantageously be provided at the point-of-care. This can reduce or remove the requirement for off-site testing and analysis, resulting in a convenient, portable and low-cost assay.

Detection of the release of protons from the amplified nucleic acid may use ISFET. It will be appreciated that as protons are released, this modulates the voltage on the ISFET, which can be detected by the ISFET when suitably biased. In some embodiments, detection by ISFET does not require modification of the ISFET gate surface area.

Alternatively, detection of the release of protons from the amplified nucleic acid may comprise optically detecting the release of protons from the amplified nucleic acid. For example, the amount of protons released may be optically detected using a pH sensitive colorimetric dye. The pH change may result from the release of protons during amplification. A change in colour of the dye after a particular time-period may be indicative of the presence of the amplified nucleic acid and thus indicate the presence of molecules of the target type in the sample.

In some embodiments amplifying any dissociated nucleic acid and detecting any amplified nucleic acid occur simultaneously by colorimetric LAMP, i.e. LAMP is carried out in the presence of a colorimetric dye, for example, a pH sensitive dye.

The sample may be a biological sample. Conveniently the biological sample may be any appropriate fluid or tissue sample obtained from a subject. For example, the biological sample may comprise at least one of urine, saliva, blood, sputum, semen, faeces, a nasal swab, tears, a vaginal swab, a rectal swab, a cervical smear, a tissue biopsy, and a urethral swab. Suitably, the biological sample is one that can be readily obtained from a subject, such as urine, saliva, blood and sputum, which the subject may be able to collect from him/herself, without the need for assistance or any invasive surgical technique. In some embodiments the sample is saliva.

In some embodiments, the sample is provided at a volume of no more than 100 pl, no more than 90μΙ, no more than 80μΙ, no more than 70μΙ, no more than 60μΙ, no more than 50μΙ, no more than 40μΙ, no more than 30μΙ, no more than 20μΙ, no more than

10μΙ, or no more than 1 μΙ. The provision of small volumes for analysis allows spare sample to be kept for other detection methods, as required.

The method of the invention may be carried out in an array format. Conveniently, this enables multiple multiplexed tests to be carried out in parallel. Thus, more than one target type may be detected.

Amplifying any dissociated nucleic acid and/or detecting any amplified nucleic acid may be carried out in a “lab-on-chip” format, for example, on a semi-conductor. The semiconductor may have amplification and/or ChemFET functionality. The method of the invention may be carried out in or on a “lab-on-chip” format, for example a semiconductor.

The aptamer-nucleic acid duplex may be provided attached to a bead. The bead may be a magnetic bead. In some embodiments the aptamer-nucleic acid duplex is provided attached to the bead by a biotin-streptavidin linkage. Other suitable attachments may be envisaged by the skilled person.

In other embodiments, the aptamer-nucleic acid duplex is provided attached to a surface of a chamber in which the method may be performed. Suitable methods of attaching the aptamer-nucleic acid duplex to the surface will be known by the skilled person.

In some embodiments, detecting any amplified nucleic acid further comprises quantifying the amount of amplified nucleic acid. Quantifying the amount of amplified nucleic acid may comprise normalising the amount of amplified nucleic acid. The amplified nucleic acid may be normalised to a no-template control.

According to a further aspect of the present invention, there is provided a method of diagnosing a subject with a condition, the method comprising:

providing an aptamer-nucleic acid duplex;

contacting a sample obtained from the subject with the aptamer-nucleic acid duplex, wherein the aptamer is capable of selectively dissociating from the nucleic acid to selectively bind to a molecule of a target type in the sample, wherein the target type is associated with the condition;

amplifying any dissociated nucleic acid; detecting any amplified nucleic acid; and using the detected result to indicate whether or not the subject has the condition.

The skilled person will be aware of suitable conditions for diagnosis which are associated with a molecule of a target type. The condition may be an infection, a disease, or a biological state. By “infection” it will be understood that the subject is infected with a microorganism such as a bacterium, a fungi, a virus, an algae, archaea or protozoa. The infection may be associated with a pathogenic phenotype (i.e. symptoms of infection), or conversely, the infection may not have a phenotype.

Suitable diseases for diagnosis may include, but not be limited to cancer, cardiovascular and/or autoimmune diseases.

In some embodiments the condition is selected from obesity or anorexia. Hence, in some embodiments the condition is selected from obesity or anorexia, and the target type is selected from one or more of leptin, ghrelin, CCK, PYY and GLP-1.

In some embodiments the condition is selected from obesity or anorexia, and the target type comprises leptin.

As described herein, the detected amplified nucleic acid is proportional to the number of molecules of a target type present in the sample. Thus, the amount of detected amplified nucleic acid may be compared to a predetermined threshold or reference “normal” level of the level/amount of the number of molecules of a target type in order to indicate whether or not the subject has the condition.

According to a further aspect of the present invention, there is provided a method of facilitating the determination of lifestyle change of a subject displaying a weightassociated condition, the method comprising:

providing an aptamer-nucleic acid duplex;

contacting a sample obtained from the subject with the aptamer-nucleic acid duplex, wherein the aptamer is capable of selectively dissociating from the nucleic acid to selectively bind to a molecule of a target type in the sample; amplifying any dissociated nucleic acid; and detecting any amplified nucleic acid,

- wherein the determination of whether or not to change the subject’s lifestyle is based upon the amount of detected amplified nucleic acid.

The weight associated condition may be obesity, anorexia or bulimia. In some embodiments the weight-associated condition is selected from obesity or anorexia.

In embodiments the weight associated condition is obesity. In such embodiments, the molecule of the target type may comprise one or more gut hormones. The gut hormone may comprise leptin. .

A lifestyle change may comprise diet or weight loss therapy. Without wishing to be bound by theory, the present inventors believe that relative modifications in leptin levels in a subject, for example, a decrease in leptin levels, are associated with future weight loss. Hence, if the subject is already on a diet or weight loss therapy, the amount of detected amplified nucleic acid may be used to determine whether or not the diet or weight loss therapy is likely to be successful and thus whether or not to continue with the diet or weight loss therapy.

Also provided is a system for detecting molecules of a target type in a sample, the system comprising:

a chamber for receiving the sample, the chamber comprising an aptamernucleic acid duplex, wherein the aptamer is capable of selectively dissociating from the nucleic acid to bind to a molecule of the target type in the sample, the system further comprising an amplification implement capable of amplifying the dissociated nucleic acid and a detection implement capable of detecting the amplified nucleic acid.

The aptamer-nucleic acid duplex may be provided in the chamber as a dry (for example, lyophilised), gel or liquid formulation.

The system may comprise a semi-conductor, which may form the base of the system. The chamber may be etched into the semi-conductor. Suitable methods of etching will be known to the skilled person, for example, using a photoresist or silicon nitride mask to provide a pattern for etching. Alternatively, the chamber may be positioned on top of the semi-conductor. For example, the chamber may be glued on top of the semiconductor.

The semi-conductor may comprise an integral ChemFET sensor. In some embodiments the semi-conductor comprises an integral ISFET sensor.

The system may further comprise a heater for heating the sample, advantageously during amplification. Advantageously, this heats the sample to one or more required temperatures to enable thermal amplification to be carried out. The device may also comprise one or more temperature sensor(s). The one or more temperature sensor(s) may be configured to control the set temperature of the heater, depending upon the temperature sensed.

The system may comprise a magnet.

In some embodiments the system comprises a plurality of chambers, each for receiving a sample. The same or different aptamer-nucleic duplexes may be provided in each chamber. In some embodiments the system comprises two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 chambers, each for receiving at least a portion of a sample. In this way, the system can be used to carry out multiple multiplexed tests to be carried out in parallel. The system may have a corresponding number of amplification and detection implements to the number of chambers. Multiple sensors, heaters, magnets and/or temperature sensors may accordingly be integrated into the semi-conductor.

The aptamer-nucleic acid duplex may be attached to a bead in the chamber. It will be appreciated that the attachment does not limit the dissociation of the nucleic acid. The bead may be a magnetic bead. In some embodiments the aptamer-nucleic acid duplex is attached to the bead by a biotin-streptavidin linkage. Other suitable attachments may be envisaged by the skilled person. It will be appreciated that the selective binding of the aptamer to a molecule of the target type and the resulting selective dissociation of the nucleic acid can occur in the chamber. Hence, for the purpose of the present invention, the chamber will herein be known as the dissociation chamber.

In other embodiments, the aptamer-nucleic acid duplex is attached to a surface of the chamber. Suitable methods of attaching the aptamer-nucleic acid duplex to the surface will be known by the skilled person.

In embodiments where the aptamer-nucleic acid duplex is attached to a surface of the chamber, this ensures that when the nucleic acid is dissociated from the aptamer, the nucleic acid is free to move while the aptamer remains bound to the component of the chamber. The nucleic acid is thus free to be amplified and detected without interference from the aptamer and aptamer-molecule duplex.

In embodiments where the aptamer-nucleic acid duplex is attached to a magnetic bead, the chamber may further comprise a magnet, for example, at the base of the chamber. In this way when the nucleic acid is dissociated from the aptamer, the nucleic acid is free to be amplified and detected without interference from the aptamer and aptamer-molecule duplex, since, the aptamer remains bound to the magnetic bead in the chamber.

The amplification and/or detection implement may be integral to the chamber. Alternatively, the amplification and/or detection implement may be located in a separate chamber to the chamber for receiving the sample. The separate chambers may be in fluid communication with each other, for example, via a channel. The channel(s) and/or chamber(s) may be microfluidic.

Hence, dissociation, amplification and detection may be carried out in the same, or separate chambers. Amplification and detection may be carried out in the same chamber.

Where dissociation is carried out in a separate chamber to amplification and detection, the dissociation chamber may be disposable. Conveniently, the amplification and detection chamber(s) can then be reused.

In some embodiments, the entire system is disposable.

In embodiments comprising a dissociation chamber and an amplification and detection chamber, the transfer of the dissociated nucleic acid from the dissociation chamber to the amplification and detection chamber may be by capillary action. Alternatively, the transfer of the dissociated nucleic acid from the dissociation chamber to the amplification and detection chamber may be by gravitational, mechanical or microelectromechanical action.

The detection implement may comprise an integral optical sensor for carrying out optical detection of any amplified nucleic acid. The optical sensor may be a CMOS or a CCD sensor. Alternatively, the system may be configured to be read by another device comprising an optical sensor in order to carry out optical detection of the amount of any amplified nucleic acid in the detection chamber.

The system may be mains power, battery or USB operated. In some embodiments the system is battery or USB operated. This advantageously removes the need for a mains power source, allowing the system to be operated by a user at the point of care, in potentially remote locations.

In some embodiments the system comprises a wireless communicator which is capable of communicating wirelessly. The wireless communicator may be capable of communicating the detected result to a wireless receiver on another device, for example a smart phone, tablet or computer. In this way the detected result may be communicated to and interpreted and/or visualised on the other device. Conveniently, this enables remote operation, and, when required, further analysis by, for example, a healthcare professional.

It will be understood that any statements made herein in relation to the first aspect of the invention will equally apply to the other aspects of the invention, and vice versa, unless stated otherwise.

Brief Description of the Figures

Figure 1 shows a top-down view of a system (without a surface cover) in accordance with an embodiment of the invention;

Figure 2 shows a top-down view of a system in accordance with an embodiment of the invention;

Figure 3 shows a flow diagram illustrating a method in accordance with an embodiment of the invention;

Figure 4 shows a flow diagram illustrating a method in accordance with another embodiment of the invention;

Figure 5 shows plots of amplification signal over cycles for the method of Figure 4; and Figure 6 shows a schematic diagram of a method for the detection of molecules of a target type in a sample in accordance with an embodiment of the present invention.

Detailed Description

Figure 1 illustrates a system 2 in accordance with an embodiment of the invention. The system comprises three microfluidic chambers 4 that each individually hold a sample volume of Ο.δμΙ. As the skilled person will appreciate however, there is no limitation to the number of microfluidic chambers within the system. Hence, in other embodiments the system comprises a plurality of microfluidic chambers so that a multiplex assay can be performed. In this way a plurality of samples can be tested, either for multiple tests for detecting the same molecule (for example, to enable the generation of an average result, i.e. to enable the statistical calculation of the significance of the results), or for detecting various different molecules at the same time. This provides a microfluidic cartridge in a simple to operate “lab on chip” format.

The cartridge of the embodiment of Figure 1 is to be considered as self-contained; in that prior to application of a sample it is substantially liquid free and is considered as dry. The only fluid prior to application of the liquid sample which is present in this cartridge is a gas, in this embodiment air.

Each microfluidic chamber is connected to an input port 6 via a microfluidic channel 8, each channel connected to and in fluid communication with an input port. A sample can be provided at the input port 6. It will be appreciated that upon provision of the sample into the input port 6, when the sample is in liquid form it will travel downstream via the microfluidic channel 8 to the microfluidic chamber 4, without the need for further elution buffer (i.e. the sample can be taken direct from a subject and provided into the cartridge). Advantageously, the sample, when provided in liquid form, does not require any manipulation before being provided to the cartridge. However, if the sample is not initially provided in liquid form, it can be eluted into the microfluidic channel 8 by the addition of an elution buffer such as PBS to the input port 6. In the present embodiment, only one input port 6 is provided, the input port being connected to each microfluidic channel 8. This enables multiple tests, either the same or different, to be carried out on the one sample. Alternatively, multiple input ports may be provided, each input port separately connected to a separate microfluidic channel.

The base of the cartridge is formed by a semiconductor substrate 10, in this embodiment. The microfluidic channels 8and chambers 4, 12 are positioned on top of the semiconductor substrate, in this embodiment by being secured by adhesive.

It will be understood that each chamber comprises a base and at least two walls defining the lumen of the chamber. The base of each microfluidic chamber is formed by the semiconductor substrate, and further comprises a magnet (not shown) integral to the semiconductor base. The magnet may be an electromagnet. Within each microfluidic chamber are magnetic nanobeads (not shown), each nanobead attached to an aptamer-nucleic acid duplex. The duplex-magnetic bead conjugates are advantageously provided as a dried formulation to enable long-term storage at ambient conditions.

Alternatively, the beads may be provided in the microfluidic chamber in a liquid buffer (i.e. in a wet formulation), for example PBS. Hence, each microfluidic chamber may be known as a dissociation chamber.

Specifically, each magnetic nanobead is coupled to a streptavidin tag. Each aptamer comprises a biotinylated tag. This enables attachment of the aptamer (which is also loosely bound to the nucleic acid) to the nanobead by a streptavidin-biotin linkage. This provides a strong binding affinity between the aptamer and the bead and fast binding kinetics. Suitable streptavidin nanobeads are available commercially, for example, Streptavidin-coupled Dynabeads (ThermoFisher Scientific).

Various protocols will be known by the skilled person for attaching biotinylated molecules to streptavidin coupled beads. Briefly, commercially-available streptavidincoupled beads are washed in a liquid buffer, for example PBS. An equal volume of biotinylated aptamer in the same buffer is added to the washed magnetic beads at up to 2-fold excess the binding capacity of the magnetic beads. The beads and aptamer are gently rotated together at room temperature for 10 minutes. After this, the aptamermagnetic bead conjugates are separated from free biotinylated aptamer by placing the reaction mixture in a vessel on a magnet for around 2-3 minutes. The free biotinylated aptamer can be washed away, for example, with PBS, and upon removal of the vessel from the magnet the aptamer-magnetic bead conjugates are washed 2-3 times in wash buffer, before being dried and inserted into each microchamber. In the system of Figure 1, the aptamer-nucleic acid duplex is attached to the beads before being placed in the microfluidic chamber.

Each of the aptamer and the nucleic acid are single-stranded DNA, each DNA sequence being complementary to the other so that the aptamer and the nucleic acid are loosely bound to each other by complementary base pair bonding (hydrogen bonds) to form a duplex.

Downstream of each microfluidic chamber 4, either directly connected to or via a further microfluidic channel 8, is a further microfluidic chamber 12, termed the amplification and detection chamber. As before, the base of each amplification and detection chamber is the semi-conductor having an integral ISFET sensor, heater and temperature sensor. The gate of the ISFET sensor forms the luminal base of the amplification and detection chamber. ISFET sensors of this nature are commercially available, for example from DNAelectronics. Advantageously, the ISFETs of this system do not require modification of the ISFET gate surface area.

Each amplification and detection chamber 12 contains lyophilised reagents (i.e. in a dry formulation) for amplification by LAMP. In other embodiments the reagents for amplification may be provided in a wet or gel formulation. The reagents include a plurality of primers which are capable of hybridising to the dissociated nucleic acid. In the present embodiment, four primers are provided, but as the skilled person will appreciate, six primers could alternatively be provided. Various mixtures of suitable LAMP reagents are available commercially, including, but not limited to, the WarmStart LAMP kit (New England BioLabs Inc.) and can be readily selected by a person skilled in the art.

Other than the input port 6, which is open to receive sample, it will be understood that the cartridge is sealed; i.e. each chamber and channel is self-contained within the cartridge, and is only in fluid communication with the inlet port. In Figure 1, the surface of the cartridge (not shown) is opaque and acts as the seal.

The cartridge of Figure 1 is provided in the form of a USB stick 14. Hence, the ISFET sensor, heater and temperature sensor are configured to be powered by electricity provided via a standard USB port, for example on a PC.

Figure 2 illustrates another system 20 in accordance with an embodiment of the invention. As described for Figure 1, the system of Figure 2 comprises a self-contained cartridge having one input port 6, at which a sample can be provided. The base of the cartridge comprises an integral magnet (not shown). In some embodiments, the magnet is an electromagnet. Integral ISFET sensors are provided in the base beneath the amplification chamber, together with a heater and temperature sensor (not shown). A processed data/pixel readout port is provided at one end of the system so that in this embodiment the cartridge forms a self-contained USB stick 14. In use, the detected information is provided via the USB stick to a USB port of a PC. The information can then be converted into filtered digital information by a program on the PC, as detailed for the system of Figure 1. As per Figure 1, the ISFET sensors, heater and temperature sensor are configured to be powered by electricity provided by a standard USB port, for example on a PC.

In contrast to the system of Figure 1, however, the system of Figure 2 comprises one dissociation chamber 4 and one amplification chamber 12. As per the system of Figure 1, the chambers and input port are connected via microfluidic channels. Flow of the sample and any reagents between the chambers, input port and microfluidic channels is, in this embodiment, microelectromechanically controlled 22. Other suitable forms of flow control can be envisaged by the skilled person.

Provided within the dissociation chamber are magnetic nanobeads attached to the aptamer as described for Figure 1, except that the nanobeads are provided in a liquid buffer, in this embodiment PBS, rather than being a dry formulation.

In this embodiment the amplification chamber contains LAMP amplification reagents as described for Figure 1, except that the reagents are provided in a liquid rather than a dry formulation.

Integral to the base of the cartridge of Figure 2 is a processing chip 24 for the processing of the proton data received in use from the amplification chamber from the ISFET sensors. The chip is linked to the USB stick 14 for the transfer of information to, for example, a PC.

In use, as illustrated by the flow diagram demonstrating a method in accordance with an embodiment of the invention, in Figure 2, a 1.8μΙ saliva sample obtained from a subject (other suitable samples and volumes can be envisaged by the skilled person) is provided by a user onto the input port of, for example, the cartridge of Figure 1. As the sample is in liquid form, the sample travels downstream by capillary action from the input port via the microfluidic channels into each of the three microfluidic dissociation chambers. In each chamber there is provided the aptamer-nucleic acid duplex, in this embodiment, as described for Figure 1, conjugated via a biotin-streptavidin linkage to magnetic nanobeads. In this embodiment, the molecules of a target type to be detected in the sample are leptin molecules. The aptamer is thus capable of selectively binding to a leptin molecule.

In the present embodiment, aptamers were selected prior to provision in accordance with the aptamer selection method of Ashley and Li (2012). Other suitable methods for selecting an aptamer which is capable of binding to a molecule of a target type will be known to the skilled person.

Once the saliva sample is in each microfluidic dissociation chamber, the liquid form of the sample acts as a buffer to reconstitute the aptamer-nucleic acid duplex conjugated magnetic nanobeads. The aptamer-nucleic acid duplex conjugated magnetic nanobeads are magnetised to the base of each chamber by the magnet. Once an aptamer-nucleic acid duplex contacts a molecule of the target type (in this embodiment, leptin), the aptamer selectively dissociates from the nucleic acid and selectively binds to the leptin molecule. This leaves dissociated nucleic acid moving freely through the sample in the microfluidic dissociation chamber, while the aptamer-molecule duplex remains conjugated to the magnetic bead and thus immobilised on the base of each dissociation chamber. In this embodiment the dissociated nucleic acid is singlestranded DNA, but other forms of nucleic acid would be suitable.

Conveniently, contact of the duplex and the molecule of the target type, in this instance leptin, occurs at room temperature. In other embodiments contact can occur at a temperature above room temperature, for example by heating of the dissociation chamber by a heater internal or external to the cartridge.

Since the aptamer-molecule duplex remains immobilised on the base of the chamber by the magnet, only the dissociated nucleic acid, and not the aptamer-molecule duplex, travels further downstream in the sample to the amplification and detection chamber.

Once within the amplification and detection chamber, the portion of the liquid sample which travelled downstream with the dissociated nucleic acid reconstitutes the lyophilised LAMP reagents, including the four primers, in the chamber.

If necessary, to assist the travel of the dissociated nucleic acid and/or the reconstitution of the reagents in the amplification and detection chamber, the dissociated nucleic acid can be eluted by an elution buffer, either added at the input port or via an internal elution chamber connected to the microfluidic channel and/or dissociation chamber. As previously mentioned, a buffer can also be added to the sample at the input port, if the sample is dry.

After reconstitution of the lyophilised LAMP reagents, the cartridge is connected to a power source, in this embodiment, a USB slot. This provides power to the heater, ISFET and temperature sensor.

It will be appreciated that the cartridge may be connected to a power source prior to reconstitution of the lyophilised LAMP reagents.

In this embodiment the heater is set to heat to a specific temperature of approximately 60-65°C for 30 minutes. This time period is sufficient to provide enough amplification of the dissociated nucleic acid for subsequent detection. The temperature sensor operates on a feedback mechanism to maintain this temperature, so when the specific temperature is reached, the temperature sensor sends a signal to the heater to stop heating. Conversely, when the temperature falls below this specific range, the temperature sensor detects this temperature and sends a signal to the heater to begin heating again.

At the required temperature, the LAMP reagents and primers function to amplify the dissociated nucleic acid. As the skilled person will appreciate, the dissociated nucleic acid is amplified by the generation of complementary nucleic acid strands from the extension of a bound primer. The complementary nucleic strands are formed from the incorporation of nucleotides (one of the LAMP reagents) into a forming strand. During extension, protons (hydrogen ions H⁺) are released as nucleotides are incorporated into the strand. As the protons are released, the voltage on the ISFET gate is modulated, which accordingly can be detected by the ISFET when suitably biased. This change in gate voltage can be used detect the amount of amplified nucleic acid. Hence, the release of protons is used to detect the amount of amplified nucleic acid at the same time as amplification.

In this embodiment, as the gate voltage changes, the detected information is provided, for example, to a PC via a USB port, and converted into filtered digital information by a program on the PC. The digital information is compared to a predetermined threshold value or reference level, which in the present embodiment represents a range of healthy levels of leptin.

The digital information is then visualised in real-time on the PC via a graphic user interface. The digital information is presented as a high, low or average level, as compared to threshold or reference level. In the present embodiment, directed to the detection of leptin, this information can assist a subject in making more informed and healthy lifestyle choices. For example, if the sample obtained from the subject has a high leptin value, then the subject may be directed to, for example one or more of, drink more water, drink less alcohol, ingest less caffeine, fat and/or sugar, increase their exercise and/or reduce their calorie intake. Conversely, if the sample obtained from the subject has a low leptin value, then the subject may be directed to, for example, reducing their exercise and/or increasing their calorie intake. Alternatively, if the subject is already on a diet or weight loss therapy, then a low leptin value may be indicative of predicted weight loss of the subject. As such, if the sample obtained from the subject has a low leptin value, for example, relative to pre-diet or weight loss therapy values, then the subject may be directed to, for example, continue on their diet or weight loss therapy. If the sample obtained from the subject has an average leptin value, i.e. equivalent to the threshold/reference value, then this may be confirmation that the subject has a healthy lifestyle and direct them to maintain their present lifestyle choices.

Alternatively, the digital information may be visualised in real-time on the PC via a graphic user interface in the form of a plot of the mV of signal produced and the time of the reaction in seconds.

Suitable alternative embodiments of the method and/or system of Figures 1, 2 or 3 will be readily envisaged by the skilled person. For example, instead of the cartridge being substantially dry prior to application of the sample, the cartridge may further comprise a buffer chamber comprising elution buffer connected to, but temporarily sealed from the microfluidic channel or the microfluidic chamber. In use, upon provision of the sample to the cartridge, the seal between the buffer chamber and the microfluidic channel/chamber can be broken, for example, by the opening of an electrically powered valve, or the actuation of an electrically powered pump provided within the buffer chamber, so that the elution buffer travels into and downstream the microfluidic channel and/or chamber.

An electrically powered pump may alternatively or in addition be provided at the input port, dissociation chamber and/or amplification and detection chambers to assist in the movement of sample downstream.

A heater may be provided which is capable of heating the dissociation chamber.

Regardless of whether the elution buffer is provided into the input port, or within a buffer chamber, in use the elution buffer advantageously elutes the sample, and, in use, the dissociated nucleic acid, downstream. This helps to separate the dissociated nucleic acid further from the magnetic-bead bound aptamer, enabling the dissociated nucleic acid to be transported to the amplification and detection chamber for subsequent amplification and detection without the potential for interference from the aptamer and/or molecule.

The microfluidic dissociation chamber and the amplification/detection chamber may be the same chamber. Hence, amplification, dissociation and detection can occur all in one chamber.

In the present embodiment, the elution buffer comprises PBS. Other suitable buffers can be envisaged by the skilled person, for example, the buffer may comprise one or more of Tris-HCI, KCI and MgCI₂.

The heater may be set to operate for up to 60 minutes.

Rather than being powered by USB, the cartridge may be battery or mains power operated. The cartridge may thus contain an integral graphic user interface, which displays the results of the method, and means for converting the information from the ISFET. A “high” level of the molecule of the target type may be simply indicated by a red light; a “low” level by an amber or yellow light and an average level by a green light. The light may be in the form of an LED. Alternatively, the level of the molecule of the target type may simply be indicated by a “yes” or a “no; wherein a “yes” indicates that the level of the molecule is too high, whereas a “no” indicates that the level of the molecule is acceptable.

Alternatively, the cartridge may be battery or mains power operated, and contain a wireless communicator which is capable of communicating wirelessly. The wireless communicator may be capable of communicating the information regarding the gate voltage change from the ISFET to a wireless receiver on another device, for example a smart phone, tablet or computer.

Instead of reagents for LAMP, the amplification and detection chamber may contain the necessary reagents for qPCR, for example, PCR primers, nucleotides, and Taq polymerase. These reagents may be lyophilised. Suitable primers, nucleotides and polymerases are well known to the skilled person, and can thus be selected and provided at a suitable amount in the amplification chamber. It will be appreciated that where amplification is by qPCR, the heater and the temperature sensor will function to operate at a number of different temperatures. An example of a standard PCR protocol is as follows: first the nucleic acid is optionally exposed to a temperature that is high enough (about 95 °C) to denature the nucleic acid by separating the two strands (if the nucleic acid is double-stranded). The temperature is then reduced (to about 55 °C) to allow annealing (i.e. binding) of primers to the single nucleic acid strands. Then the temperature is increased (to about 72 °C) so that a polymerase enzyme starts synthesizing a copy strand by extension of the primer bound on the template nucleic acid. Ideally, at each stage of the amplification, the number of nucleic acid copies doubles.

When the cartridge is self-contained, the surface of the cartridge may be transparent or translucent.

Alternatively, the surface of the cartridge may be open, with the cartridge having a separate lid which may be placed on the cartridge to seal the contents and to selfcontain the cartridge. The lid may be transparent, translucent or opaque, as required. In embodiments comprising a lid, an input port may not be required, as each sample can simply be placed into the chamber and then the lid closed.

In some embodiments, the cartridge does not contain an ISFET sensor. Instead, a colorimetric dye may be provided as one of the LAMP reagents, the dye being sensitive to pH. Hence, in use, as protons are released during amplification, this leads to a change in the pH of the sample in the amplification and detection chamber, leading to a change in colour of the colorimetric dye. The change in colour can be visualised manually by eye, or, alternatively, the cartridge can be inserted into another device containing an optical sensor such as a CMOS or a CCD sensor. The sensor can then read the change in colour. The information regarding the change of colour from the sensor can then be converted into filtered digital information by a program on the device. The digital information may be compared to a predetermined threshold value or reference level. The digital information can then be visualised on the device via a graphic user interface, or alternatively transferred to a PC for visualisation. The digital information is presented as a high, low or average level, as compared to threshold or reference level. As before, this information may be presented as a “yes” or a “no”. Alternatively, the camera of a smart phone may be used to read the change in colour, the information regarding this being converted into filtered digital information by an app on the phone.

Figure 4 shows a method in accordance with another embodiment of the invention. The method shown in Figure 4 is comparable to the method of Figure 3, except that amplification is by qPCR rather than LAMP. Hence, in the system used in this embodiment, qPCR reagents are provided in the amplification and detection chamber, rather than LAMP reagents. In addition, qPCR unlabelled primers and fluorescent probes (in this embodiment in the form of TaqMan probes) were provided not as separate reagents in the amplification and detection chamber, but instead pre-bound to the nucleic acid of the aptamer-nucleic acid duplex. In other embodiments the qPCR primers and/or probes may be provided separately in the amplification and detection chamber.

Three samples were provided, each sample being a test sample of elution buffer containing a predetermined leptin concentration of 0.01ng/ml, 0.1ng/ml or 1ng/ml. Accordingly, in the system for use in this method, the system contains three separate input ports, each input port being connected to a separate microfluidic chamber via a microfluidic channel.

In contrast to the method of Figure 3, the aptamer-nucleic acid duplex is not conjugated to magnetic beads, but is instead immobilised on a surface of the microfluidic chamber via a thiol group. Other suitable forms of immobilisation will be known by the skilled person. Hence, in the present embodiment, a magnet is not required.

In contrast to the method of Figure 3, detection of the amount of amplified nucleic acid is not by an ISFET sensor, but instead by optical detection, in this embodiment in the form of a CMOS optical sensor, provided by a device separate to the system. In alternative embodiments detection of the amount of amplified nucleic acid is by an ISFET sensor or by an optical sensor integral to the cartridge. Therefore, the apparatus for use with this embodiment does not contain an ISFET sensor, and is inserted into the device having the CMOS optical sensor, to enable the real-time reading of fluorescence, during use. To enable the reading of any fluorescence signal, the cartridge is sealed with a transparent lid.

As for the method of Figure 3, after dissociation, the dissociated nucleic acid, in the form of single-stranded DNA, travels downstream in the sample to an amplification and detection chamber. Then the temperature is increased by the heater (to about 72 °C) so that a polymerase enzyme present in the reagents starts synthesizing a copy strand by extension of the primer bound on the template nucleic acid. When the polymerase reaches a TaqMan probe, it cleaves the probe, separating the dye from the quencher, resulting in fluorescence. Collectively, this is known as one PCR cycle. In the method, the cycle was repeated for a further 39 cycles, for a total of 40 cycles.

In embodiments where the probes and/or primers are provided separately in the amplification/detection chamber (i.e. not pre-bound to the nucleic acid), the dissociated nucleic acid, once in the amplification and detection chamber, is heated by the heater to a temperature that is high enough (about 95 °C) for the fluorescent dye of the 5’ end of the TaqMan probe to be quenched by the NFQ on the 3’ end. The temperature is lowered (if necessary) to a temperature suitable to allow annealing of qPCR primers and the probe to the single stranded nucleic acid DNA. In the present embodiment this temperature is approximately 55 °C. After this the temperature is increased by the heater (to about 72°C) as described above, so that a polymerase enzyme present in the reagents starts synthesizing a copy strand by extension of the primer bound on the template nucleic acid.

The fluorescence emitted each cycle was detected by the CMOS sensor, and this information plotted on a graph, as shown in Figure 5.

Figure 5 shows the amplification and quantification curves of the amplified nucleic acid as a measure of dissociated nucleic acid displaced by leptin in accordance with the method of Figure 4. The amount of amplified nucleic acid was measured by fluorescence emitted from the TaqMan probes, as detailed above. As Figure 5 shows, the amplification curves directly correlate to the concentration of leptin (0.01ng/ml, 0.1ng/ml and 1ng/ml) in the sample. Hence, the higher the concentration of leptin in the sample, the greater the amount of dissociated nucleic acid and thus fluorescence is generated at an earlier cycle stage. Amplification and quantification was possible even at very low concentrations of leptin, as shown by the curve demonstrating amplification of the dissociated nucleic acid when the sample contained 0.01ng/ml of leptin. Thus, the present method is highly sensitive yet convenient. It will be appreciated that this information can be converted into filtered digital information by a program on the device having the CMOS sensor. The digital information may be compared to a predetermined threshold value or reference level. The digital information can then be visualised on the device via a graphic user interface, or alternatively transferred to a PC for visualisation. The digital information can be presented as a high, low or average level, as compared to threshold or reference level. As described above, the information can alternatively be presented as a “yes” or a “no”. Alternatively, the camera of a smart phone may be used to read the change in colour, the information regarding this being converted into filtered digital information by an app on the phone.

With reference to Figure 6, a method of detection according to an embodiment of the invention is illustrated. In this embodiment, the molecules of a target type to be detected are leptin molecules. The aptamer is thus capable of selectively binding to a leptin molecule. In the present embodiment, aptamers were selected prior to provision in accordance with the aptamer selection method of Ashley and Li 2012. The aptamernucleic acid duplex is attached to a magnetic bead via a streptavidin-biotin linkage, as discussed above. Each of the aptamer and the nucleic acid are single-stranded DNA, each DNA sequence being complementary to the other so that, as shown in Figure 6A, the aptamer and the nucleic acid are loosely bound to each other to form a duplex.

As shown in Figure 6B, upon the provision of a 10μΙ saliva sample containing leptin, the sample containing the leptin contacts the aptamer-nucleic acid duplex. Since the aptamer has a higher affinity for the leptin than for its complementary nucleic acid, the aptamer selectively dissociates from the nucleic acid to instead selectively bind to the leptin. This leaves the dissociated nucleic acid unbound and free for amplification and quantification. As described above, the single stranded DNA nucleic acid can then be amplified by PCR or LAMP and the amplified DNA detected by any suitable means as described above. From the start to the finish of the method, the present method may be completed in around 60 minutes or less.

The present invention thus provides a novel and innovative method, and associated system, for detecting any molecule conveniently, portably and simply, but particularly, for detecting molecules associated with a subject’s lifestyle, the results of which can be used to direct the subject in altering their lifestyle accordingly.

References

Ashley J. and Li S. (2012). Three dimensional Selection of Leptin Aptamers using capillary electrophoresis and Implications for Clone Validation. Analytical Biochem. 434: 146-152.

Pan HT, Guo J and Su ZQ (2014). Advances in understanding the interrelations between leptin resistance and obesity. Phys. And Behav. 130:157-169.

Tomita N., Mori Y., Kanda H and Notomi T (2008). Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products. Nat. Protocols. 3:877-882.

CLAIMS:

Claims (22)

1. A method of detecting molecules of a target type in a sample, the method comprising:

providing an aptamer-nucleic acid duplex;

contacting the sample with the aptamer-nucleic acid duplex, wherein the aptamer is capable of selectively dissociating from the nucleic acid to selectively bind to a molecule of the target type in the sample;

amplifying any dissociated nucleic acid;

detecting any amplified nucleic acid; and using the detected result to indicate the presence of molecules of the target type and/or quantify an amount of molecules of the target type.

2. A method according to claim 1, wherein detecting the amount of amplified nucleic acid comprises detecting a release of protons from the amplified nucleic acid.

3. A method according to claim 1 or 2, wherein detecting any amplified nucleic acid comprises detecting any amplified nucleic acid using a chemical field-effect transistor (ChemFET).

4. A method according to claim 3, wherein the ChemFET comprises an ion-sensitive field-effect transistor (ISFET).

5. A method according to claim 1 or 2, wherein detecting any amplified nucleic acid comprises optically detecting any amplified nucleic acid.

6. A method according to claim 5, wherein optically detecting any amplified dissociated nucleic acid uses a CMOS or CCD sensor.

7. A method according to any preceding claim, wherein amplifying any dissociated nucleic acid and detecting any amplified nucleic acid occur at substantially the same time.

8. A method according to any preceding claim, wherein amplifying any dissociated nucleic acid comprises thermally amplifying any dissociated nucleic acid.

9. A method according to claim 8, wherein thermally amplifying any dissociated nucleic acid comprises qPCR or LAMP.

10. A method according to any preceding claim, wherein the nucleic acid comprises or consists of DNA, RNA, or combinations thereof.

11. A method according to claim 10, wherein the nucleic acid comprises or consists of DNA.

12. A method according to any preceding claim, wherein the sample comprises at least one of urine, saliva, blood, sputum, semen, faeces, a nasal swab, tears, a vaginal swab, a rectal swab, a cervical smear, a tissue biopsy, and a urethral swab.

13. A method according to any preceding claim, wherein the aptamer comprises or consists of a nucleic acid.

14. A method according to any preceding claim, wherein the target type comprises a peptide.

15. A method according to any preceding claim, wherein the target type comprises a hormone.

16. A method according to claim 15 or claim 16, wherein the target type comprises one or more of leptin, ghrelin, CCK, PYY and GLP-1.

17. A system for detecting molecules of a target type in a sample, the system comprising:

a chamber for receiving the sample, the chamber comprising an aptamernucleic acid duplex, wherein the aptamer is capable of selectively dissociating from the nucleic acid to bind to a molecule of the target type in the sample, the system further comprising an amplification unit capable of amplifying the dissociated nucleic acid and a detection unit capable of detecting the amplified nucleic acid.

18. A system according to claim 17, wherein the detection unit comprises one or more ChemFET sensors.

19. A system according to claim 18, wherein the or each ChemFET sensor is an ISFET sensor.

20. A system according to any one of claims 17-19, and comprising one or more magnetic beads to which the aptamer-nucleic acid duplex is attached.

21. A system according to any one of claims 17-20, wherein the amplification and detection units are integral to the chamber.

22. A system according to any one of claims 17-21, wherein said amplification unit comprises a heater and one or more microfluidic channels.

Intellectual Property Office

Application No: GB1800629.6