GB2602597A

GB2602597A - Method and assay kit to detect an analyte

Info

Publication number: GB2602597A
Application number: GB2204182.6A
Authority: GB
Inventors: Sabyah Abdullah
Original assignee: Rightangled Ltd
Current assignee: Rightangled Ltd
Priority date: 2016-07-14
Filing date: 2016-07-14
Publication date: 2022-07-06
Also published as: GB202204182D0

Abstract

The invention provides a method of detecting the presence of an analyte in a sample comprising the steps of; a) contacting the sample with a first capture ligand which is conjugated to a first metal nanoparticle whereby the first capture ligand binds to the analyte, b) contacting the first capture ligand bound to the analyte with a second different capture ligand, which is conjugated to a second metal nanoparticle, whereby the second capture ligand also binds to the analyte and c) detecting the binding of the metal nanoparticles to the analyte. Also disclosed is a kit for use in detecting an analyte comprising a first and second capture ligand conjugated to metal nanoparticles. Preferably the analyte is cell-free fetal DNA or RNA, the capture ligands are nucleic acid probes and the metal nanoparticle is a gold nanoparticle. The nucleic acid probes may be comprised of locked nucleic acids. The detection of the analyte can be used to diagnose any genetic or pathological conditions typified by the presence of a specific DNA sequence or to determine the gender of a fetus. Also disclosed is a computer implemented method to aid diagnosis.

Description

METHOD AND ASSAY KIT TO DETECT AN ANALYTE

This invention relates to methods and assay kits for detecting an analyte. Methods and assay kits for determining the presence of a specific DNA sequence, including identifying the gender of a fetus, are also provided. Apparatuses for biological sample collection and processing, for example for use in an assay, arc also provided. A computer implemented method of providing information relating to a diagnosis, including the diagnosis of fetus gender, is also provided.

in vitro diagnostic devices are typically used to determine the presence of an analyte in a biological sample. These devices can be used in qualitative and/or quantitative analysis and may be adapted for use as a home test kit or a point of care test kits. These types of test kits can be operated by an unskilled user either in a home setting or in a clinic in the presence of a healthcare professional. Pregnancy test kits are one of the most common home test kits, which are capable of being carried out by a user who has no specific training in assay kits or the interpretation of assay results. Therefore, the advantages of developing a home test kit include the ease of operation of the device, the lack of requirement for training on how to use the kit and the privacy afforded by the ability to carry out the test at home.

Lateral flow test kits are traditionally imtnunochromotographic assays. They are used to rapidly identify the presence or absence of a particular analyte in a sample. The assay is usually enclosed inside a plastic housing as a test strip, or dipstick, with specialised regions to receive, process and view the sample. The test strip is often fixed to an inert backing. Samples do not usually require pre-treatment. Lateral flow assays can also be integrated with electronic systems, for example, to indicate a quantity of the analyte in a readable format.

A lateral flow test strip usually comprises four separate regions; a sample pad, a conjugate or reagent pad, a reaction membrane and an adsorbent pad or waste reservoir.

The sample pad is usually made of cellulose and/or glass fibres. The sample pad receives the sample, which is typically a biological liquid sample such as urine. As the sample comes into contact with the sample pad, the assay is activated. The liquid sample migrates through the sample pad, moving laterally across the length of the test strip. The movement of the sample is intended to be homogenous and continuous. Sample pads may also contain reagents which are required for pre-treatment of the sample, including separation, buffering, filtering or cleaning. They may also be known as plasma separation membranes.

The conjugate or reagent pad is where the conjugated ligands are located which have been selected for their ability to specifically bind to the analyte of interest. The ligands are conjugated to a label. They are immobilised by temporary adsorption to the conjugate pad by a bond which is broken upon contact with a liquid. The porosity, structure and composition of the material the conjugate pad is formed from can be selected for particular desired properties including sensitivity and release rates. Typically, conjugate pads are formed from cellulose, glass fibres and/or various polyesters. The labelled ligand binds to the analyte of interest in the liquid sample and is transported laterally onwards through the conjugate pad matrix. Typically, the ligands are polyclonal antibodies which bind specifically to the analyte of interest. Aptamcrs may also be used as ligands. The ligands can he labelled by a number of conjugate labels including enzymes, coloured dyes, fluorescent dyes or nanoparticles.

The reaction membrane is usually a nitrocellulose membrane. Membranes are graded and can be selected for a required sensitivity, binding properties and support. A test line and a control line are located on the reaction membrane. The liquid sample containing unbound analyte and analyte bound to the labelled ligand will be adsorbed onto the membrane. The liquid moves across the reaction membrane via capillary action.

The absorbent pad or reservoir acts as a sponge to adsorb excess liquid sample and regulates the continuous flow of the liquid across the test strip. The properties of the absorbent pad will determine the adsorption rate across the test strip.

There are two types of lateral flow assays; sandwich and competitive. Sandwich assays which use antibodies are most common. In a sandwich assay, the test line contains immobilised primary antibodies which are specific for the analyte of interest. Secondary antibodies which are specific for the conjugated labelled antibodies located in the conjugate pad are immobilised at Ihe control line. The sample is applied to the sample pad where it travels to and contacts the conjugate pad. The analyte of interest in the liquid sample binds to the conjugated labelled antibody here and travels to the test line. At the test line, the primary antibody binds to the analyte which may or may not be bound to the conjugated labelled antibody. If the analyte is bound to the conjugated labelled antibody from the conjugate pad, the analyte is said to be "sandwiched" between the primary antibody and the conjugated labelled antibody. The liquid sample then contacts the control line, where the secondary antibodies bind any excess remaining conjugated labelled antibody. Any excess liquid sample will travel on to the absorbent pad. The increased number of labelled antibodies at the test line in close proximity to one another will result in a visible change (such as a colour change), whilst a visible change at the control line indicates that the test is working correctly. The intensity, for example, of the colour change at the test line may be interpreted to indicate a quantitative value of the analyte.

Competitive assays are often used for smaller molecules which are unable to bind two binding ligands, such as antibodies, simultaneously. The sample pad and conjugate pad function in the same way as in a sandwich assay, hut the test line contains the analyte of interest which is immobilised to the test line. As the sample liquid reaches the test line, the immobilised analyte binds the conjugated labelled ligand. If the analyte is at low levels in the initial liquid sample, a larger number of the conjugated labelled ligands will bind to the test line. The analytes are said to compete for the binding site on the conjugated labelled ligand. A subsequent increase in, for example, colour intensity at the test line indicates the absence or low levels of the analyte in the liquid sample. This can also be measured quantitatively. A secondary ligand specific for the conjugated labelled ligand is immobilised to the control line to indicate that the test is working correctly.

Lateral flow tests usually have a very long shelf life and remain stable over a variety of environmental conditions. The sample volume required is relatively small, for example, a typical pregnancy test requires 125111 of urine or 100RI of blood. Lateral flow tests can also he multiplex. These tests can detect the presence of more than once analyte simultaneously.

Recently, lateral flow tests have been developed for use with metal nanoparticles, particularly gold nanoparticles. Gold nanoparticles are used to label ligands which bind to the analyte of interest. Typically, colloidal gold nanoparticles are used as they are relatively inert and can produce highly spherical particles. These particles are usually charged which ensures they have an affinity for protein molecules. Proteins can be adsorbed against or covalently attached to the surface of gold particles.

The advantage of using gold nanoparticle to label proteins is that when there is an increase in number and density of the gold nanoparticles, for example, when the conjugated protein binds to the test line in a lateral flow assay, this results in a visible colour change. When gold nanoparticles are exposed to light, the oscillating electromagnetic field of light causes a concerted oscillation of the free electrons in the gold. This oscillation is in resonance with the frequency of visible light which are known as surface plasmons. The surface plasrnon resonance of the nanoparticles dictate the absorption of light of a particular spectra. Small gold nanoparticles (approximately 30nm in diameter) cause light to be absorbed at approximately 450nm and light is reflected at approximately 700nm. This results in a visible red colour.

The optical properties of gold nanoparticles change when the nanoparticles come into close contact with one another and aggregate. The electrons on each particle surface become delocalised and are shared with nanoparticles in close proximity. The surface plasmon resonance shifts to a lower energy and the absorption peak for light shifts Therefore, the collection of gold particles in close proximity will result in a colour change. Therefore, when conjugated to a ligand, the binding of that li2and to a particular area such as a test strip will bring the gold particles into dose proximity and result in a visible colour change. Usually, unaggregated gold particles are red in solution. When the particles aggregate, the solution becomes blue/purple in colour.

Cell-free DNA is DNA which is not localised to the nucleus of a cell. It can be detected in circulating blood and tissues. Many pathological conditions such as cancers, heart disease and sepsis cause an increase in cell-free DNA levels. Measuring cell free DNA levels has recently been used to indicate the presence of tumour cells or underlying disease. Typically, quantitative PCR methods are used to amplify, detect and quantify the cell-free DNA of interest.

Cell-free fetal DNA is DNA from a fetus which can be found in the maternal circulation. Approximately 2 to 6% of cell-free DNA in the maternal circulation is of fetal origin. Cell-free fetal DNA fragments are often around 200bp in size. Cell free fetal DNA results from the turnover of villous tropho blast material into maternal circulation. It has recently been found that cell-free DNA can be detected from five weeks of gestation. Detecting and analysing cell-free fetal DNA can be used to diagnose various inherited disorders such as haemophilia. Duchenne's muscular dystrophy, congential adrenal hyperplasia or aneuploidy. The paternity of a child, gender and blood group can also be determined.

Detecting cell-free DNA simply requires a blood sample and is therefore classed as noninvasive. Traditional methods such as chorionic villus sampling or amniocentesis involves the removal of placental cells and amniotic fluid respectively. These are invasive techniques which pose a small risk of miscarriage.

Determining the gender of a fetus may confirm or rule out the possibility of the fetus being affected by any sex-linked disease which affects either males or females. This can be particularly useful if the parents of the child are aware that they may be carriers for a particular sex-linked disease. Being able to determine the gender and likelihood that a child will have a particular disease allows the parents and relevant health professionals to prepare and arrange for any support the child or mother may require. Determining the gender of a fetus therefore has important healthcare implications for both the fetus and the mother.

Currently, lateral flow assays have not been developed in order to detect cell-free fetal DNA. Traditionally, PCR methods have been used to amplify and detect cell-free fetal DNA.

W02014120379 discloses a lateral flow assay which utilises gold particles to label ligands which bind specifically to HIV virion proteins. This assay uses one set of gold particles conjugated to a ligand which binds specifically to an HIV virion in order to take part in a sandwich style assay. The conjugated labelled ligand is located in the conjugate pad and travels along with the liquid sample, in this case blood, to the test line in order to bind to a primary binding ligand which is immobilised on the test line. The result of the assay can then be viewed through a window. If the HIV virion protein is present, a colour shift from red to blue can be observed through the window. It is emphasised that the volume of sample required and the extremely low levels of virions present (1 virion in 50u1 of blood) in the sample present a significant problem for these types of assays in the diagnostics field.

The current applicants have unexpectedly found that utilising two sets of labelled gold nanoparticles in a lateral flow assay results in a highly sensitive point-of-care assay which can be operated by an unskilled user at home or in the clinic Furthermore, the volume of sample required is approximately two drops of blood (approximately 1000), which is a volume of blood easily obtained in a non-clinical setting.

The inventors have realised that labelling specific DNA probes with gold nanoparticles provides many unexpected advantages. The invention uses two different sets of gold nanoparticles (also known as AuNPs), one set conjugated to one set of DNA probes made of forward and/or reverse primers, and a second set which are coated with a linking molecule, such as streptavidin, and immobilised at low density at the test line by physioisorption. A second set of forward and/or reverse primers are also used which are complementary for a region of DNA which is adjacent to the regions to which the AuNP conjugated primers are complementary to. This second set of probes contain a linking molecule at the 3' end, such as biotin. The DNA probes are selected based on an intended target sequence, for example, a portion of the SRY gene on the Y chromosome. The specific binding of the probes to the DNA sequence has a much higher affinity than any antibody interaction. An advantage of using the Y chromosome is that the mother will not have her own Y chromosome present.

The invention may also use two different sets of gold nanoparticles (also known as AuNPs), one set conjugated to one DNA probe, and another set being conjugated to another different DNA probe. The DNA probes are selected based on an intended target sequence, for example, a portion of the SRY gene on the Y chromosome.

Conjugating DNA probes to one set of gold nanoparticles for use in a lateral flow kit is known. However, the current invention is dependent on the interaction of two different DNA probes conjugated to either gold nanoparticles or a linking molecule, such as biotin, which are capable of binding to adjacent regions on the same sequence. Therefore, binding of the probes to the sequence is highly specific and more accurate when compared to traditional assays which only rely on one probe to bind to the sequence of interest. Binding of, for example, a biotinylateci set of probes with streptavidin coated AuNPs on the test line will cause the gold nanoparticles to come into very close proximity with one another, and therefore aggregate. This would likely result in a clearer, more definitive result which is achieved over a shorter amount of time. The likelihood of unspecific binding is also extremely low when two probes are used. Furthermore, the use of two sets of probes allows for both the sense and antisense DNA strands to be detected. This further amplifies the detection signal indicating the presence of the target double stranded DNA sequence.

Furthermore, the test kit described herein eliminates the requirement for prior amplification of DNA by PCR. Traditionally detecting the presence of a specific DNA or RNA sequence in a sample is achieved by selecting primers, or probes, which are specific for that sequence and performing a PCR reaction in order to amplify the DNA and observe the presence of the DNA using traditional methods such as gel electrophoresis. The amplification of DNA is particularly required for cell-free DNA, which could be present in levels as low as >10 fmol/ul of blood. Furthermore, carrying out a PCR method requires significant expertise which is not usually possessed by the average user of a home test kit. The expense and handling of reagents required for PCR must also be considered, along with the time required to prepare the samples and run a PCR method cycle. The risk of cross-contamination with other amplified products is also possible in a laboratory setting. Therefore, an assay method which is able to detect very low levels of DNA and which can be incorporated into a home test kit has a number of advantages.

The current applicants have also developed a device which can be used to receive and process a biological fluid sample, such as blood. This can be incorporated into the body of lateral flow kit. Traditionally, lateral flow kits requiring blood as the sample liquid include a step whereby blood is removed from the subject, either by a pin prick or intravenous sampling, which is subsequently applied directly to the sample pad. Often, blood samples are also required to undergo processing and treatment prior to being administered to the sample pad, for example, plasma separation.

In particular, the current device has been developed in order to eliminate any processing or treatment steps which are required to be carried out by the user. The sharps used to harvest the blood sample are incorporated into the device, eliminating the need for separate sharps to be used with the device. Furthermore, the device has also been developed so that the sharps used to harvest the blood sample are secured away permanently once the sample has been received. This reduces any opportunity for cross-contamination or transmission of blood borne diseases.

Cultural bias towards the preference for a male child is well known in the modern world for various reasons which are not discussed here. The current inventors are aware of the need to support the expectant mother and immediate family by providing an early, accurate indication of the gender of the child and subsequent health and social care support. This may include access to a healthcare practitioner such as a doctor or nurse, a genetic counsellor, a mental health counsellor or other expectant mothers.

The current inventors have combined the newly developed lateral flow assay test kit and the sample receiving and processing device described herein with a software application. The software application can be downloaded and installed onto a hand held computing device such a smart phone. The software application enables the steps of ordering the home test kit following validation of personal details, providing instructions and support as to how to carry out the test kit, processing of the results and providing a diagnosis. The test kit can be ordered to arrive at the user's home or at a designated clinic, where the test can be carried out under supervision or by the user alone. The interpretation of the results will only be possible with the use of the software application, which will involve the user taking a picture of the test kit window. The software will then compare the picture to existing pictures to determine a diagnosis, for example, whether the fetus is male or female. This diagnosis is communicated to the user via the software application and the result may be recorded in a secured database for any future reference.

Depending on the diagnosis, the software application then provides communication links with various individuals who can provide support and advice to the patient or expectant mother such as a healthcare practitioner, a genetic counsellor, a mental health counsellor or other users within the community and further afield. The software application will also contain information relating to the specific condition that the device is intended to diagnose. For example, the software will contain information relating to pregnancy and fetal development if the device is used to detect the Y chromosome in cell free fetal DNA. This tailored healthcare approach provides unique support to the expectant mother and her unborn child.

A first aspect of the invention provides a method of detecting the presence of an analyte in a sample comprising the steps of a) contacting the sample with a first capture ligand which is conjugated to a first metal nanoparticle and a second different capture ligand which is conjugated to a first linking molecule, whereby the first and second capture ligands bind to the analyte in the sample, thereby forming a first metal nanoparticle-first capture ligandsecond capture ligand-first linking molecule-analyte complex, b) providing on a substrate a second linking molecule conjugated to a second metal nanoparticle, c) contacting the complex with the second linking molecule conjugated to the second metal nanoparticle allowing the first and second linking molecules to bind to one another, and d) detecting the binding of the metal nanoparticles to the analyte.

Also provided herein is an assay kit for use in detecting an analyte in a sample comprising a) a first capture ligand specific for the analyte which is conjugated to a first metal nanoparticle, and b) a second different capture ligand which is conjugated to a first linking molecule, and c) a second metal nanoparticle which is conjugated to a second linking molecule.

Typically the substrate is any surface to which the second linking molecule conjugated to the second metal nanoparticle can be adsorbed or physisorbed onto. The substrate may be contacted via any anchoring molecule generally known in the art.

Typically the method is carried out using the assay kit disclosed herein.

Typically, the analyte is a nucleic acid, such as DNA, typically cell-free DNA. The cell-free DNA may be from a benign or malignant tumour cell. The subject providing the sample may be suspected of having a disease, such as cancer, an inflammatory disease, sepsis or heart disease. The cell free-DNA may be present in the biological sample at levels between 20 and 4000 ng/ml of whole blood.

Typically the cell-free DNA is fetal in origin. The test may be carried out at any time during pregnancy. Typically the test is carried out between five and seven weeks of gestation, typically at least at seven weeks of gestation. The sample is typically a whole blood sample from a pregnant subject.

The sample is typically whole blood, plasma or serum. The sample may be urine, cerebrospinal fluid, lymph fluid, tissue or tumour tissue, or any other biological sample.

Typically the sample is at least 40u1 to 100u1 in volume.

Typically the sample is received by the apparatus disclosed herein and subsequently disposed onto the sample pad of the test kit. The sample may also be collected by finger prick with a lancet. or sharp including a needle or venepunclure and may be pipetted or dropped into the sample pad or into the apparatus. The sample may have been collected and stored prior to use in the method or kit. A capillary tube may be used to collect and transfer the sample to a plasma separation membrane before the sample pad. The sample pad may comprise a plasma separation membrane.

Typically the kit is a lateral flow test comprising the first and second capture ligands as defined above. The lateral flow test typically comprises a test strip which comprises a plasma separation membrane, a sample pad, a conjugate pad, a reaction membrane and a reservoir pad. The lateral now test typically comprises a test line and a control line. Typically, the materials selected for each component part of the test strip is selected for the required adsorptive and wicking properties. The kit is capable of being stored at room temperature for an extended amount of time.

The lateral flow test may be housed inside an inert housing, such as plastic, whereby the test strip is completely encased within and unable to be touched by the subject or user. The lateral flow test may comprises a window through which the test line and the control line are visible to the naked eye.

Typically the sample contacts a plasma separation membrane prior to contacting the sample pad. The plasma separation membrane may contain additional reagents required to process the sample. The plasma separation membrane is typically a GFX Vividum membrane. The sample pad may contain additional reagents required for DNA purification and denaturation.

Typically, the sample is administered to capillary tube which releases the sample onto a plasma separation membrane where the sample filtrates through the membrane and is collected directly onto a pre-treated sample pad. The sample pad is pre-treated with proteinase K and SDS to purify the sample and aid dissociation of any bound proteins to the target free cell DNA. It is also treated with NaOH to increase the plI of the sample and to denature the two strands of the target DNA in the sample. The user waits for a predetermined amount of time, such as one to ten minutes, typically five minutes before a chase buffer is released to neutralise the pH in the sample pad. The chase buffer may be released by the user operating the device. Typically the sample pad size and material type is selected to retain the plasma until the chase buffer is required to be released.

Typically, the capture ligands are nucleic acid probes. The capture ligands may also be antibodies or fragments thereof or aptamers.

Typically, the first capture ligand is an antisense nucleic acid sequence, or a forward primer. Typically, the second capture ligand is a different antisense nucleic acid sequence, or a forward primer. Typically, the second capture ligand is a sense nucleic acid sequence, or a reverse primer. Typically, the second capture ligand is a different sense nucleic acid sequence, or a reverse primer. The first capture ligand may be known as probe 1 and the second capture ligand may be known as probe 2.

Probes 1 and 2 may be selected to be complementary to different specific portions of DNA, for example, a portion of the SRY gene of the Y chromosome. Typically, probes 1 and 2 may represent a continuous antisense strand which is complementary to a sense strand of the target DNA sequence. Typically, probes 1 and 2 may represent a continuous sense strand which is complementary to an antisense strand of the target DNA sequence.

Typically, one set of probes 1 and 2 are used (for example, two forward primers). Typically. one set of probes 1 and 2 are used (for example, two reverse primers).

Step (a) of the method may further comprise contacting the sample with a third capture nucleic acid sequence conjugated to a metal nanoparticle wherein step (b) further comprises contacting the sample with a fourth capture nucleic acid sequence conjugated to a linking molecule.

Typically, the third capture ligand is a sense nucleic acid sequence, or a reverse primer. Typically, the fourth capture ligand is a different sense nucleic acid sequence, or a reverse primer. Typically, the third capture ligand is an antisense nucleic acid sequence, or a forward primer. Typically, the fourth capture ligand is a different antisense nucleic acid sequence, or a forward primer. The third capture ligand may be known as probe 1 and the fourth capture ligand may be known as probe 2.

The kit may also further comprise a third nucleic acid probe which is conjugated to a metal nanoparticle, and a fourth different nucleic acid probe which is conjugated to a linking molecule. Typically, the third nucleic acid probe is a sense nucleic acid sequence, or a reverse primer. Typically, the fourth capture ligand is a different sense nucleic acid sequence, or a reverse primer. Typically, the third capture ligand is an antisense nucleic acid sequence, or a forward primer. Typically, the fourth capture ligand is a different antisense nucleic acid sequence, or a forward primer. The third capture ligand may be known as probe 1 and the fourth capture ligand may he known as probe 2.

Typically, both sets of probes 1 and 2 (forward/forward and reverse/reverse primers) are used in order to amplify the signal further. In this event, there will be four separate primers bound to both an antisense strand and a sense strand of the target DNA. Typically, any linking molecule conjugated to the fourth nucleic acid probe is capable of binding to the first linking molecule.

Typically, the first linking molecule is biotin and the second and/or fourth linking molecule is streptavidin.

Typically, the first and/or third capture ligand (for example. a forward and/or a reverse primer) is conjugated to a metal nanoparticle at the 5' end. These may be known as set A metal nanoparticles. Typically, the linking molecule conjugated to the second and/or fourth capture ligand (for example, a forward and/or a reverse primer which is different to the first capture ligand) is biotin, therefore the second and/or fourth capture ligand (a nucleic acid probe) is biotinylated at the 3' end.

The linking molecule may be any linking molecule known in the art.

Typically, multiple first and/or third and second and/or fourth nucleic acid probes are conjugated to individual metal nanoparticics. Typically, multiple first and/or third and second and/or fourth nucleic acid probes are biotinylated and dispensed on the conjugate pad. These probes may represent different regions on both strands of the target analyte and hence provide an increased sensitivity limit in detecting the target sequence.

Typically, the second linking molecule conjugated to the second and/or fourth metal nanoparticle is streptavidin or avidin. Typically, the second metal and/or fourth nanoparticle is coated with streptavidin molecules. These metal nanoparticle conjugates may be known as set B. Typically, the first (also known as set A) and second (also known as set B) metal nanoparticles are gold nanoparticles. Typically, the first set (A) is conjugated to probe 1, while the second set (B) is streptavidin coated and immobilised on the test line.

The metal nanoparticle may also be silver or copper. Typically, the gold nanoparticle is approximately 40 nm in diameter. The particles may the same or different.

Typically, for example, the first capture ligand-first metal nanoparticle conjugate and the second different capture ligand-first linking molecule conjugate are temporarily immobilised to the conjugate pad by methods well known in the art. The temporary bond is releasable upon contact with a liquid. For example, when the liquid sample containing the DNA sequence of interest reaches the conjugate pad, the first capture ligand-first metal nanoparticle conjugate hybridises with the complementary target DNA sequence and forms a first metal nanoparticle-first capture ligand-target DNA-second different capture ligand-first linking molecule conjugate complex. This may also he known as a metal nanoparticle-probe 1-target DNA-probe 2-biotin complex. This complex may then travel to the test line whereby the immobilised streptavidin coated nanoparticles bind to the biotin conjugated second capture ligand. The complex is now immobilised at the test line.

Typically, the second set of streptavidin coated gold nanoparticles (set B) are immobilised to the test line by methods well known in the art, including the use of streptavidin and biotin and/or physisorption.

Typically, the method comprises the use of a control capture ligand, typically a sense nucleic acid sequence which is complementary to the anti sense sequence of probe I, which is used to capture first and/or third capture ligands which have not bound to the analyte of interest. The control capture ligand may be known as probe 3. Typically, a sense sequence to probe 1 (probe 3) is immobilised to the control line by methods well known in the art, including the use of streptavidin and biotin. Therefore, any remaining nucleic acid probe 1 sequences conjugated to nanoparticles (set A) in the liquid sample after travelling past the test strip will be immobilised at the control line.

Typically, the kit contains a control capture ligand. typically a sense nucleic acid sequence, for example, a sense sequence to probe 1 (probe 3) which is immobilised to a control line by methods well known in the art, including the use of streptavidin and biotin and/or phy sisorption.

Typically, the binding of the hybridised complex to the second set of immobilised nanoparticles is detected as a colour change of the test line which is visible to the naked eye. Before the kit is used or the method is undertaken, the test line is typically slightly red in colour and the control line is colourless. Typically, if gold nanoparticles are used, the colour of the test line may intensify and change from red to blue/purple (if the analayte of interest is present) or a colour change will not occur and the test line will remain slightly red. If the test kit has worked correctly, the control line will also change colour, for example. from colourless to red. If the test kit has not worked correctly, the control line will not change colour, for example, it will remain colourless. This will indicate that the kit did not work correctly and that the result cannot be relied upon. The intensity of the colour may also be detected to indicate a quantitative value for the analytc.

The colour change will result from the close proximity of the metal nanoparticles to one another, also known as aggregation. If the capture ligands are nucleic acid probes, this can be described as DNA induced aggregation.

Any kit disclosed herein may additionally comprise one or more of instructions for using the kit, substrate, a buffer, label, a preservative or a control.

Typically, the nucleic acid probes are comprised of locked nucleic acids. Locked nucleic acids reduce background noise and have a high thermal stability when forming duplexes. This enables a high sensitivity of target detection to be achieved.

Typically, the detection of the binding of the analyte is used to determine paternity or blood group type or is used to diagnose haemophilia. Duchenne muscular dystrophy or congenital adrenal hyperplasia. Typically the test line indicates qualitative data, such as a "negative" or "positive" result, "negative" indicating the absence of a disease and "positive" indicating the presence of a disease. For example, a negative result is typically associated with the test line remaining a faint red colour, and a positive result is typically associated with an intensified red colour at the test line or a change of colour from red to purple.

Typically, the detection of the binding of the analyte is used to determine the gender of a fetus. For example, a colour change of the test line from light red to intense red to blue/purple indicates that the Y chromosome is present and therefore that the fetus is male. An absence of a colour change of the test line value indicates that the Y chromosome is absent and therefore that the fetus is female.

Typically, the capture ligands, for example forward and reverse primers, are selected to detect the Y chromosome. Typically, a portion of the SRY gene on the Y chromosome or a portion of the DYS 14 or the DAZ sequence on the Y chromosome is selected. The portion is typically 50bp in length or shorter.

Any method or kit disclosed herein may comprise multiple target probes directed towards detecting the presence of and/or measuring the quantitative amount of one or more analytes simultaneously. is

Typically the descriptive information regarding the diagnosis is not available to the user on the kit or in the instructions for the kit. The colour change intensity or the colour change to blue/purple can only he interpreted via the software application.

Another aspect of the invention provides a method of detecting the presence of an analyte in a sample comprising the steps of a) contacting the sample with a first capture ligand which is conjugated to a first metal nanoparticle whereby the first capture ligand binds to the analyte in the sample; b) contacting the first capture ligand bound to the analyte with a second different capture ligand which is conjugated to a second metal nanoparticle whereby the second capture ligand also binds to the analyte in the sample; and c) detecting the binding of the metal nanoparticics to the analyte.

Also provided herein is an assay kit for use in detecting an analyte in a sample comprising; a) a first capture ligand specific for the analyte which is conjugated to a first metal nanoparticle and; h) a second different capture ligand which is also specific for the analyte which is conjugated to a second metal nanoparticle.

Typically, the analyte is a nucleic acid, such as DNA, typically cell-free DNA. The cell-free DNA may be from a benign or malignant tumour cell. The subject providing the sample may be suspected of having a disease, such as cancer, an inflammatory disease, sepsis or heart disease. The cell free-DNA may he present in the biological sample at levels between 20 and 4000 ng/m1 of whole blood.

The sample is typically whole blood, plasma or serum. The sample may be urine, cerebrospinal fluid, lymph fluid, tissue or tumour tissue, or any other biological sample. Typically the sample is at least 40u1 to 100u1 in volume.

Typically the method is carried out using the assay kit disclosed herein.

Typically the sample is received by the apparatus disclosed herein and subsequently disposed onto the sample pad of the test kit. The sample may also be collected by finger prick with a lancet or sharp including a needle or venepuncture and may be pipetted or dropped into the sample pad or into the apparatus. The sample may have been collected and stored prior to use in the method or kit. A capillary tube may be used to collect and transfer the sample to a plasma separation membrane before the sample pad. The sample pad may comprise a plasma separation membrane.

Typically the kit is a lateral flow test comprising the first and second capture ligands as defined above. The lateral flow test typically comprises a test strip which comprises a plasma separation membrane, a sample pad, a conjugate pad, a reaction membrane and a reservoir pad. The lateral flow test typically comprises a test line and a control line. Typically, the materials selected for each component part of the test strip is selected for the required adsorptive and wicking properties. The kit is capable of being stored at room temperature for an extended amount of time.

The lateral flow test may be housed inside an inert housing, such as plastic, whereby the test strip is completely encased within and unable to be touched by the subject or user. The lateral flow test may comprises a window through which the test line and the control line are visible to the naked eye Typically the sample contacts a plasma separation membrane prior to contacting the sample pad. The plasma separation membrane may contain additional reagents required to process the sample. The plasma separation membrane is typically a GFX Vividum membrane. The sample pad may contain additional reagents required for DNA purification and denaturation.

Typically, the sample is administered to capillary tube which releases the sample onto a plasma separation membrane where the sample filtrates through the membrane and is collected directly onto a pre-treated sample pad. The sample pad is pre-treated with proteinase K and SDS to purify the sample and aid dissociation of any bound proteins to the target free cell DNA. It is also treated with NaOH to increase the pH of the sample and to denature the two strands of the target DNA in the sample. The user waits for a predetermined amount of time, such as one to ten minutes, typically five minutes before a chase buffer is released to neutralise the pH in the sample pad. The chase buffer may be released by the user operating the device. Typically the sample pad size and material type is selected to retain the plasma until the chase buffer is required to be released.

Typically, the capture ligands are nucleic acid probes. The capture ligands may also be antibodies or fragments thereof or aptamers. Typically, the first capture ligand is an antisense nucleic acid sequence, or a forward primer. Typically, the second capture li2and is typically a sense nucleic acid sequence, or a reverse primer. The forward and reverse primers are selected to be complementary to a specific portion of DNA, for example, a portion of the SRY gene of the Y chromosome. Typically the forward and reverse primers represent a continuous antisense strand which is complementary to the target DNA sequence.

Typically the nucleic acid probes are conjugated to the gold nanoparticle at the 5' end of the probe. Typically multiple nucleic acid probes are conjugated to individual gold nanoparticles.

Typically, the first and second metal nanoparticles are gold nanoparticles. The metal nanoparticle may also be silver or copper. Typically, the gold nanoparticle is approximately 40 mu in diameter. The particles may the same or different.

Typically the forward primer-gold nanoparticle conjugate is temporarily immobilised to the conjugate pad by methods well known in the art. The temporary bond is releasable upon contact with a liquid. For example, when the liquid sample containing the DNA sequence of interest reaches the conjugate pad, the forward primer-gold nanoparticle conjugate hybridises with the anti-sense DNA sequence and forms a DNA-forward primer-gold nanoparticle complex. This complex travels to the test line whereby the immobilised reverse-primer-gold nanoparticle conjugate hybridises with the sense DNA sequence. The complex is now immobilised at the test line.

Typically, a reverse primer-gold nanoparticle conjugate is immobilised to the test line by methods well known in the art, including the use of streptavidin and biotin and physisorption.

Typically, the method comprises the use of a control capture ligand, typically an anti-sense nucleic acid sequence, which is used to capture first capture ligands which have not bound to the analyte of interest. Typically, a forward primer-gold nanoparticle conjugate is immobilised to the control line by methods well known in the art, including the use of streptavidin and biotin. If any remaining DNA sequences which are complementary to the forward primer are still remaining in the liquid sample after travelling past the test strip, being either hybridised to a forward primer-gold nanoparticle conjugate (a DNA-forward primer-gold nanoparticle complex) or free and unbound, they will be immobilised at the control line.

Typically, the kit contains a control capture ligand, typically an anti-sense nucleic acid sequence, for example, a forward primer-gold nanoparticle conjugate which is immobilised to a control line by methods well known in die art, including the use of streptavidin and biotin.

Typically, the binding of the first capture ligand and first nanoparticle to the second ligand and second nanoparticle is detected as a colour change of the test line which is visible to the naked eye. Before the kit has been activated, the test line is typically faint red in colour and the control line is colourless. Typically, if gold nanoparticles are used, the colour change of the test line may intensify and change from faint red to intense red to blue/purple (if the analyte of interest is present) or a colour change will not occur aml the test line will remain slightly red. If the test kit has worked correctly, the control line will also change colour, for example, from colourless to red. If the test kit has not worked correctly, the control line will not change colour, for example, it will remain colourless. This will indicate that the kit did not work correctly and that the result cannot be relied upon. The intensity of the colour may also be detected to indicate a quantitative value for the analyte.

The colour change will result from the close proximity of the metal nanoparticles to one another, also known as aggregation. If the capture ligands are nucleic acid probes, this can be described as DNA cross-linked aggregation.

Typically, the detection of the binding of the analyte is used to determine paternity or blood group type or is used to diagnose haemophilia, Duchenne muscular dystrophy or congenital adrenal hyperplasia. Typically the test line indicates qualitative data, such as a "negative" or "positive" result, "negative" indicating the absence of a disease and "positive" indicating the presence of a disease. For example, a negative result is typically associated with the test line remaining a faint red colour, and a positive result is typically associated with an intensified red colour at the test line or a change of colour from red to purple.

Typically, the detection of the binding of the analyte is used to determine the gender of a fetus. For example, a colour change of the test line from light redto intense red to blue/purple indicates that the Y chromosome is present and therefore that the fetus is male. An absence of a colour change of the test line value indicates that the Y chromosome is absent and therefore that. the fetus is female.

Typically, the capture ligands, for example forward and reverse primers, are selected to detect the Y chromosome. Typically, a portion of the SRY gene on the Y chromosome or a portion of the DY514 sequence on the Y chromosome is selected. The portion is typically 50bp in length or shorter.

Any method or kit disclosed herein may comprise multiple target probes directed towards detecting the presence of and/or measuring the quantitative amount of one or more analytes simultaneously.

Typically the descriptive information regarding the diagnosis is not available to the user on the kit or in the instructions for the kit. The colour change intensity or the colour change to blue/purple can only be interpreted via the software application.

Also provided herein is an apparatus for processing a biological sample, comprising a housing, the first surface of which comprises a well which extends into the housing, a hinged lid connected to the first surface of die housing. the lid comprising an outer and an inner surface, the inner surface forming a protrusion which extends into the well when the hinged lid is urged towards the first surface of the housing, the boundaries of the well being formed by the inner surface of the lid and the outer surface of the housing, a sharp within the well, a tubular member comprising a proximal and a distal end, being connected to a resilient member, said resilient member being fixed to the first surface of the housing in the well, an aperture in the first surface of the housing in the well, a membrane which can be accessed through the aperture in the first surface of the housing in the well, whereby urging of the inner surface of the lid towards the first surface of the housing causes the protrusion to enter the well and contact the tubular member, thereby compressing the msilient member and forcing the tubular member towards the membrane, and whereby the distal end of the tubular member contacts the membrane when the lid closes the well.

Typically the apparatus additionally comprises a diagnostic test strip in fluid communication with the membrane. Typically the apparatus is for use with or is incorporated in a lateral flow assay.

Typically, when the apparatus is not in use and has not yet been activated, the well is closed, for example, by a temporary membrane which must be pierced or removed before use. Once the device has been used to collect a biological sample, the lid is closed and secured which seals away the sharp and reduces the risk of injury.

Typically, the lid is prevented from being urged away from the first surface of the housing once the tubular member has contacted the membrane. Typically, the lid is connected to an edge of the first surface at the boundary of one edge of the well.

Typically, the apparatus additionally comprising a releasable chase buffer. Typically, the buffer is located within a buffer chamber. There may be two buffer chambers opposite one another and adjacent to the membrane. The buffer chamber may not be in fluid communication with the membrane until the chase buffer is required to be in fluid communication with the membrane, for example, five minutes after the sample has contacted the membrane.

The buffer chamber may comprise a wall adjacent to the membrane which is pierceable by a sharp or lancet. The lancet or sharp may be connected to a member or button which is operable by the user from the exterior of the device. The lancet or sharp may extend into the buffer chamber and terminate before contacting the wall of the buffer chamber adjacent to the membrane. The button may be urged towards the housing by the user depressing the button. This may force the sharp or lancet to pierce the wall of the buffer chamber adjacent to the membrane, causing the buffer to come into contact with the membrane.

In another embodiment, the wall of the buffer chamber adjacent to the membrane may commise an aperture which is closed by a hinged door. The hinged door may be connected to one edge of the aperture in the wall of the buffer chamber. The exterior wall of the housing may form the lateral wall of the buffer chamber. The exterior wall of the housing may be deformable. When the exterior wall is forced inwards towards the interior of the housing, this may cause a decrease in the volume of the buffer chamber and a compression of the liquid buffer inside the buffer chamber. The increase in the liquid pressure may he adequate enough to urge the hinged door outwards to open the aperture and allow the buffer to exit the buffer chamber. The liquid may then contact the membrane.

Typically the chase buffer may be released upon the user depressing a button located on the exterior of the apparatus. Typically the user is required to wait five minutes following the harvesting of the liquid sample before releasing the chase buffer.

Typically, the liquid sample, for example a blood sample, is mixed with the chase buffer after passing through a plasma separation membrane. In another embodiment, the chase buffer is released from a chamber and mixes with the blood sample outside of the chamber once the sample has passed through a plasma separation membrane, for example, five minutes after the sample has passed through a plasma separation membrane.

Typically, the lid is prevented from being urged away from the first surface of the housing once the tubular member has contacted the membrane and upon the user activating the release of the chase buffer. Typically the prevention of the lid being urged away from the first surface of the housing once the tubular member has contacted the membrane is activated by the act of the user depressing a button to cause the release of the chase buffer. The button may, for example, break a seal on a portion of the apparatus containing the chase buffer. This releases the buffer.

In another embodiment, the lid is prevented from being urged away from the first surface of the housing once the tubular member has contacted the membrane upon the user depressing a button located on the exterior of the apparatus, and a further depression of the same button causes the chase buffer to be released.

In another embodiment, the chase buffer is released upon the user depressing a button located on the exterior of the apparatus, and a further depression of a different button causes the lid to be prevented from being urged away from the first surface of the housing once the tubular member has contacted the membrane.

In another embodiment, the chase buffer is released automatically one to ten, two to eight or typically five minutes after the user has urged the inner surface of the lid towards the first surface of the housing causes the protrusion to enter the well and contact the tubular member, thereby compressing the resilient member and forcing the tubular member towards the membrane, and whereby the distal end of the tubular member contacts the membrane when the lid closes the well.

Typically, the depression or urging of a button forces the chase buffer to exit a chamber.

Typically, the biological sample is whole blood, plasma or serum. The sample may be urine, cerebrospinal fluid, lymph fluid, tissue or tumour tissue, or any other biological sample.

Typically the sample is between 40u1 to 100u1 in volume.

Typically, the biological sample is a liquid and travels through the apparatus via capillary action.

Typically, the sharp is a sterile needle or lancet.

Typically, the collection vessel is a capillary tube. Typically, the capillary tube has a volume of 40u1 to 100u1.

Typically, the resilient member is a coiled spring.

Typically, the membrane is a plasma separation membrane. Typically, once the liquid sample has passed through the plasma separation membrane it passes onto a sample pad.

Also provided herein is a computer implemented method for providing information relating to a diagnosis, comprising the steps of capturing an image of an assay result obtained by any method or kit disclosed herein using a camera comparing the image to existing images on a database to determine a diagnostic slate transmitting information conveying the diagnostic state to a user; and providing a user selectable link to initiate communication with a remote device.

Typically, a logarithmic scale is used to quantify the colour and intensity of the image when compared to existing images on a database.

Typically, the remote device is operated by a healthcare professional, a counsellor, a genetic counsellor, a community member or another user who has received information conveying a diagnostic state.

Any kit or method disclosed herein may be used to identify the presence of any specific cell free DNA sequences which correlate with a genetic or pathological condition. Typically, the diagnostic state is the gender ofa fetus. The diagnostic state could also be the absence or presence of a disease such as haemophilia. Duchenne muscular dystrophy or congenital adrenal hyperplasia. The diagnostic state could also be paternity or blood group type.

Typically, the diagnostic state is determined using any method disclosed herein or by using any assay kit disclosed herein. Typically, the assay used is any assay disclosed herein.

Typically the image is of a test line and a control line of a lateral flow assay, such as the assays disclosed herein. Typically, the diagnostic state may also indicate that the kit has worked incorrectly. The image may be indicative of the assay not working correctly, for example the control line remains colourless. The user is notified of this diagnostic state and may be prompted to repeat the assay. The assay cannot be repeated with the same device, the user may be prompted to return the kit so they can receive another one.

Typically, the camera is a smartphone camera or a personal digital assistant camera.

Typically, the method comprises the step of storing the captured image on a database.

Typically, the user will be provided with a user selectable link to information related to fetal development and pregnancy healthcare.

Typically, the computer implemented method is downloadable as a software application for use on a smartphone or a personal digital assistant or other suitable computing means.

Typically, any assay kit disclosed herein is able to be ordered using the software application. Typically, the user is prompted to enter personal details prior to ordering any assay kit disclosed herein.

Also disclosed herein is a computer readable medium storing instructions which when performed can carry out any computer implemented method disclosed herein.

Also described herein is an electronic device having a processor and a memory, the memory storing instructions which when carried out cause the processor to carry out the steps of capturing an image of an assay result obtained by any method or kit disclosed herein using a camera, comparing the image to existing images on a database to determine a diagnostic state, transmitting information conveying the diagnostic state to the user; and providing a user selectable link to initiate communication with a remote device.

Any embodiment described herein is intended to be applied to any method, kit, apparatus, software or hardware described herein. There are no restrictions on to which invention each embodiment refers to.

The invention will now be described by way of example only, with reference to die following figures: Figure 1 shows an embodiment of the lateral flow assay kit disclosed herein.

Figure 2 shows the hybridisation complex and associated probes.

Figure 3 shows a further embodiment. of the lateral flow assay kit disclosed herein.

Figure 4 shows the apparatus for processing a biological sample.

Figure 5 shows the interior of the apparatus showing the releasable buffer mechanism.

Figure 6 shows an external view of the apparatus.

Figure 7 shows a flow diagram of an embodiment of the computer implemented method.

With reference to Figure 4, there is provided an apparatus 10 for processing a biological sample, comprising a housing 12, the first surface 14 of which comprises a well 16 which extends into the housing 12, a hinged lid 18 connected to one edge of the first surface 14 of the housing 12, the lid 18 comprising an outer 20 and an inner surface 22, the inner surface 22 forming a protrusion 24 which extends into the well 16 when the hinged lid 18 is urged towards the first surface 14 of the housing 12, the boundaries of the well 16 being formed by the inner surface 22 of the lid 18 and the outer surface 20 of the housing 12, a sharp 26 within the well 16, a tubular member 28 comprising a proximal 30 and a distal 32 end, being connected to a resilient member 34, said resilient member 34 being fixed to the first surface 14 of the housing 12 in the well 16, an aperture 36 in the first surface 14 of the housing 12 in the well 16, a membrane 38 which can be accessed through the aperture 36 in the first surface 14 of the housing 12 in the well 16, whereby urging of the inner surface 22 of the lid 18 towards the first surface 14 of the housing 12 causes the protrusion 24 to enter the well 16 and contact the tubular member 28, thereby compressing the resilient member 34 and forcing the tubular member 28 towards the membrane 38, and whereby the distal end 32 of the tubular member 28 contacts the membrane 38 when the lid 18 closes the well 16.

The housing 12 is substantially cuboidal and elongate in order to accommodate the pad of a fingertip. The housing has a first surface 14, a bottom surface and four side surfaces. The first surface 14 is substantially square and flat and comprises a central well 16 which is hemispherical with an oval perimeter to accommodate the pad of a fingertip. The well 16 extends into the main body of the housing 12.

The hinged lid 18 is connected to one edge of the first surface 14 of the housing 12 allowing 1800 of rotational movement in one plane about the axis of the edge of the first surface 14. The hinged lid comprises an outer surface 20 which is substantially flat and square and an inner surface 22. The inner surface 22 is substantially flat and square except for a protrusion 24 which extends into the well 16 when the lid 18 is being urged towards full closure or is fully closed. The substantially flat area of the inner surface 22 of the lid 18 entirely covers the substantially flat surface of the first surface 14 of the housing 12 and the well 16 opening when the lid 18 is closed. The protrusion 24 is substantially cylindrical with a top face and a lateral face and there is clearance around the lateral face when the protrusion 24 extends into the well 16. The top boundary of the well 16 is formed by the substantially flat inner surface 22 of the lid 18 and the bottom boundary of the well is formed by the hemispherical outer surface 20 of the housing 12 when the lid 18 is closed. A sharp 26 is located within the well 16 in close proximity to the bottom of the well 16 and extends into the centre of the well 16.

The tubular member 28 is hollow and open at both ends and comprises a proximal 30 and a distal 32 end. The tubular member 28 is connected to a resilient member 34 midway along the shaft of the tubular member so that the proximal 30 end of the tubular member 28 is arranged to project towards the inner surface 22 of the lid 18 and the distal 32 end is arranged to project towards the bottom of the well 16. The resilient member 34 is permanently fixed to the first surface 14 of the housing 12 in close proximity to the bottom of the well 16.

The aperture 36 in the first surface 14 of the housing 12 is located at the bottom of the well 16. The membrane 38 is accessible through the aperture 36 in the first surface 14 of the housing 12 in the well 16. The aperture 36 is typically circular and has a diameter larger than the diameter of (he tubular member 28. Urging of the inner surface 22 of the lid 18 towards the first surface 14 of the housing 12 causes the protrusion 24 to enter the well 16 which contacts the proximal 30 end of the tubular member 28. The resilient member 34 is reversibly deformed by the protrusion 24 which forces the distal 32 end of the tubular member 28 towards the aperture 36.

The membrane 38 is located inside the housing 12 inferior to the well 16 and is accessible via the aperture 36. The membrane is connected to the interior of the housing. The distal end 32 of the tubular member 28 contacts the membrane 38 when the lid 18 closes the well 16. The well 16 is closed when the inner surface 22 of the lid 18 contacts and is flush against the substantially flat first surface 14 of the housing 12.

In an alternate configuration, the apparatus 10 additionally comprises a diagnostic test strip 40 in fluid communication with the membrane 38.

The diagnostic test strip 40 extends throughout the housing and is connected to the membrane 38.

In an alternate configuration, the apparatus 10 additionally comprises a releasable chase buffer which is located in a buffer chamber 42. The buffer chamber 42 is located within the housing 12 and may be in fluid communication with the membrane 38. The wall of the buffer chamber 42 adjacent to the membrane 38 may be pierceable.

The buffer chamber 42 is substantially cuboidal and holds a predetermined volume of chase buffer. The chamber has a top face, a bottom face and four side faces. One lateral side face is formed by one of the side faces of the housing. This lateral side face may comprise a button 44 or member which extends through the side face into the buffer chamber 42. The button 44 is connected to a lancet 46 which extends into the buffer chamber 42 and terminates in the buffer chamber with clearance before the side face adjacent to the membrane 38. Depression or urging of the button 44 causes the button to move laterally through the lateral side face of the housing 12 and partially into the interior of the housing whilst maintaining a water tight seal between the chamber and the lateral side face of the housing 12. The lancet also moves laterally towards the buffer chamber 42 and contacts the side face adjacent to the membrane causing rupturing of the side face adjacent to the membrane and the release of the buffer from the buffer chamber 42. The buffer can travel to the membrane 38.

Figure 5 is a simplified drawing of an alternative embodiment of the buffer chamber 42 and the membrane 38. The buffer chamber is located adjacent to the well 16 and is separated by the bottom boundary of the well 16 which is formed by the first surface 14 of the housing 12. The aperture 36 is located centrally at the bottom of the well 16 and the liquid sample 48 travels across the membrane 38.

The side wall of the buffer chamber 42 adjacent to the membrane 38 may comprise an aperture 52 which is closed by a hinged door 54. The hinged door 54 may be connected to one edge of the aperture 52 in the side wall of the buffer chamber 42 adjacent to the membrane 38. The lateral side wall of the housing 12 may be deformable. When the lateral side wall of the housing 12 is forced inwards towards the interior of the housing 12, this may cause a decrease in the volume of the buffer chamber 42 and a compression of the liquid buffer inside the buffer chamber 42. The increase in the liquid pressure may be adequate enough to urge the hinged door 54 outwards to open the aperture 52 and allow the buffer to exit the buffer chamber 42. The liquid may then contact the membrane 38.

The liquid sample 48 contacts the test strip 40 after mixing with the buffer.

Figure 7 discloses a Row diagram of an embodiment of the computer implemented method. At step 1.0, an image is captured of an assay result using a camera. At step 2.0, the image is compared to existing images on a database to determine a diagnostic state. At step 3.0, information conveying the diagnostic state is transmitted to a user. At step 4.0, the user is provided with a user selectable link to initiate communication with a remote device.

Lateral flow test kit Method and materials Hybridisation is between each strand of the target DNA with two probes binding to adjacent regions on the same complementary ssDNA causing the target to be bound with a maximum of four probes.

There are two categories of AuNPs involved:-Set A = AuNPs bound to a forward primer (sense strand) or a reverse primer (antisense strand) at the 5 'end (localised at the conjugate pad).

Set B = AuNPs are coated with streptavidin (localised at the test line) There are also two categories of probes:-Probe 1 = a forward primer (sense strand) or a reverse primer (antisense strand) are labelled with AuNPs at the 5' end (also known as Set A, localised at the conjugate pad).

Probe 2 = a forward primer (sense strand) or a reverse (antisense strand) primer are biotinylated at the 3' end (also localised at the conjugate pad).

Using two primers (forward and reverse) causes the hybridisation complex to occur on both strands, this approach doubles the amplification signal and results in a higher sensitivity for the test.

Therefore, two sets of AuNPs (40 nm or other sized diameters) are required. The conjugated probes and target SRY gene sequence on the Y chromosome are as follows:-Primer I Forward: 5'-CA CTG GTA TCC CAG CTG CTT -3' Primer 2 Forward: S.-GC TGA TCT CTG AGT TTC GCA TT-3' Target Forward: 5'-AAT GCG AAA CTC AGA GAT CAG CAA GCA OCT GGG ATA CCA GTG -3' Primer I Reverse: 5'-CTA TGA ATA TTA AGC CCA TAA -3' Primer 2 Reverse: 5'-AGA GAG ACA CUT ACC QUA CAT-3' Target Reverse: 5'-OAT ACT TAT AAT TCG GOT ATT TCT CTC TOT GCA TOG CCT GTA -3' For AuNP conjugation: Poly A tail and polyethylene glycol (PEG) used as a spacer to increase the flexibility of the sequence, a thiol group for conjugating the probes to the nanoparticles, TCEP for reducing the disulfide bonds.

Probes I conjugation to AuNPs is from 5' end, and probes 2 biotinylation is from 3'end. The two sets of AuNPs are as follows:-AuNP set A (conjugate pad): 5'-ThioMC6-A15-PEG18-(probe 1 sequence Forward) / (probe 1 sequence Reverse) -3' Immobilised AuNP set B(test line): Streptavidin coated The capture probe on the control line can be immobilised by streptavidin (or by any other conventional procedure) and the sequence is antisense to probe I Forward, also known as Probe 3, on the control line. Probe 3 is immobilised through biotinylation and bound with anchored streptavidin on the control line:-Probe 3: 5'-BIOTIN-TEGI 5-Al 5-AAG CAG CTG GGA TAC CAG TO -3' The immobilisation of the second set of gold nanoparticles on the test line can be achieved by coating the nanoparticles with streptavidin and attachment to the nitrocellulose membrane through physical adsorption.

The device (Figure 1), will carry out two consecutive steps:-First step: blood separation and DNA purification and denaturation 1. Blood sample 40-100 ttL is spotted on the plasma separation membrane which is placed over an adsorbent pad (sample pad).

2. Plasma separation takes place through a filtration membrane, such as a GFX membrane, which will yield a high quality plasma in less than 2 minutes with? 80% plasma retrieved (VIVID plasma separation membrane GFX).

3. The plasma yield will be absorbed on a sample pad that would be pre-treated with 1% SDS, 10 Rg/m1Proteinase k and 1 M NaOH; for DNA purification and denaturation.

After five minutes, another click on the device (adjacent to the sample pad) will release the chase buffer (a neutralising buffer Tris-1-IC1). This will neutralise the high pH in the sample pad. The conjugate pad will be pre-treated with a -dried" hybridisation buffer; which comprises 5X SSC buffer and 16% dextran sulfate. It will also contain the dispensed biotinylated probe 2 and the AuNPs set (A)-probe 1 conjugates.

Second step: DNA hybridisation and AuNPs target-induced aggregation 1. Once the sample (the neutralising buffer along with the plasma, which contains the target analyte) has reached the conjugate pad, the AuNPs-probe 1 and the biotinylated probe 2 are released and the probes can hybridise with the target ssDNA and form a hybridised complex (AuNP-probe 1-target-probe 2-biotin).

2. The flow of sample liquid with the conjugated AuNPs then reaches the test line, where the second set of streptavidin coated AuNPs (Set B) are immobilised.

3. At the test line, the biotin labelled probe 2 binds with streptavidin on the surface of AuNPs Set B and the hybridised complex is now captured at the test line.

4. The AuNP capturing via biotin-streptavidin interaction will cause the two sets of gold nanoparticles (A and B) to come into close proximity with each other and induce their aggregation. This will result in a change of colour at the site of immobilised nanoparticles on the test line, leading to an amplified detection reading of the target analyte.

Furthermore, at the control line, the capture probe would be immobilised and the sequence is complimentary to probe 1 (probe 1 anti-sense). Therefore, any unbound AuNP-probe 1 conjugates can be captured at the control line. These hybridisation events will cause the nanoparticles to aggregate and change colour on the test line and/or cause a redcolour on the control line.

Apparatus method Figure 4 discloses an embodiment of the apparatus for processing a biological sample. First, a user is intended to prick their finger using the incorporated sharp and a volume of blood (40 to 100u1) is collected in a capillary tube. The capillary tube is attached by a spring which ensures that the blood sample is directly applied to a plasma separation membrane once the cap is closed.

Once the cap is closed, the plasma is filtrated through the plasma separation membrane and is treated on the "pre-treated" sample pad with NaOH, Proteinase K and SDS. Then the chase buffer can he applied applied manually by the user. The user would he instructed to press a button after five minutes after closing the cap. The chase buffer may also be released automatically after five minutes. From here, the filtrated plasma sample will neutralise and then mix with the hybridisation buffer on the conjugate pad along with the conjugated first set of gold nanoparticles and biotinylated probe 2. The sample will then reach the test strip. The movement of the sample through the device is via capillary force.

The side click of the chase buffer may be incorporated within the cap (in a two level lock fashion) to reduce the risk of injury once the kit has been used.

Figure 5 discloses a simplified example of the chase buffer release mechanism.

Figure 6 discloses the hypothetical use of the device:-Step 1 -The user presses on the middle of the device to trigger the sharp and prick finger (the sharp can only be activated once).

Step 2 -Collect a drop of blood on the blood collection tip.

Step 3 -Close the cap: the act of closing the cap allows the blood collection tube to contact the plasma separation membrane which releases the blood sample onto the pad. A second click of the cap (after five minutes) then triggers the plasma sample and buffer to mix together and travel to the conjugate pad, where hybridisation takes place and then travels to the reaction pad (the test strip).

Step 4 -Wait for result (around five minutes). If the test device window reveals a colouration of the test line, the gender is male.

Statements of Invention

A method of detecting the presence of an analyte in a sample comprising the steps of:-a) contacting the sample with a first capture ligand which is conjugated to a first metal nanoparticle and a second different capture ligand which is conjugated to a first linking molecule, whereby the first and second capture ligands bind to the analyte in the sample, thereby forming a first metal nanoparticle-first capture ligand-second capture ligand-first linking molecule-analyte complex; b) providing on a substrate a second linking molecule conjugated to a second metal nanoparticle; c) contacting the complex with the second linking molecule conjugated to the second metal nanoparticle allowing the first and second linking molecules to bind to one another; and d) detecting the binding of the metal nanoparticles to the analyte.

2. An assay kit for use in detecting an analyte in a sample comprising:-a) a first capture ligand specific for the analyte which is conjugated to a first metal nanoparticle; and b) a second different capture ligand which is conjugated to a first linking molecule; and c) a second metal nanoparticle which is conjugated to a second linking molecule.

The method of clause 1 or the assay kit of clause 2 wherein die analyte is a DNA sequence, preferably cell-free DNA, more preferably cell-free fetal DNA.

The method or kit according to any previous clause wherein the analyte is an RNA sequence, preferably cell-free RNA, more preferably cell-free fetal RNA.

5. The method or kit according to any previous clause wherein the capture li2ands are nucleic acid probes.

6. The method or kit according to clause 5 wherein the first and second capture nucleic acid probes are anti-sense nucleic acid sequences located adjacent to one another on the same complementary strand to a target nucleic acid sequence.

7. The method or kit according to any preceding clause wherein the first linking molecule is biotin and wherein the second linking molecule is streptavidin.

8. The method according to clause 6 wherein step (a) further comprises contacting the sample with a third capture nucleic acid sequence conjugated to a metal nanoparticle and wherein step (b) further comprises contacting the sample with a fourth capture nucleic acid sequence conjugated to a linking molecule.

9. The kit according to clause 6 further comprising; a) a third nucleic acid probe which is conjugated to a metal nanoparticle; and h) a fourth different nucleic acid probe which is conjugated to a linking molecule.

10. The kit or method according to clauses 8 or 9 wherein the linking molecule conjugated to the fourth nucleic acid probe is capable of binding to the first linking molecule.

11. The method or kit according to any preceding clause wherein the first linking molecule is biotin and the second and/or fourth linking molecule is streptavidin.

12. The method or kit according to any of clauses 8 to II wherein the third and fourth capture nucleic acid probes are sense nucleic acid sequences located adjacent to one another on the same complementary strand to a target nucleic acid sequence.

13. The method according to any preceding clause wherein a control capture ligarid, typically a sense nucleic acid sequence which is complementary to the first nucleic acid probe sequence, is used to capture first capture ligands which have not bound to the analyte.

14. The kit according to clause 2 further comprising a control capture ligand, typically a sense nucleic acid sequence which is complementary to the first nucleic acid probe sequence.

15. The method according to any preceding clause wherein a control capture ligand, typically a sense nucleic acid sequence which is complementary to the third nucleic acid probe sequence, is used to capture third capture ligands which have not bound to the analyte.

16. The kit according to clause 2 further comprising a control capture ligand, typically a sense nucleic acid sequence which is complementary to the third nucleic acid probe sequence.

17. The method or kit according to any of clauses 5 to 16 wherein the nucleic acid probes are comprised of locked nucleic acids.

18. The method or kit according to any previous clause wherein the metal nanoputicle is a gold nanopaiticle.

19. The method or kit according to any preceding clause wherein the quantitative amount of an analyte is determined.

20. The method or kit according to any preceding clause wherein the detection of the binding of the analyte is used to diagnose any genetic or pathological conditions typified by the presence of a specific nucleic acid sequence.

21. The method or kit according to any preceding clause wherein the detection of the binding of the analyte is used to determine paternity or blood group type or is used to diagnose haemophilia, Duchenne muscular dystrophy, congenital adrenal hypeiplasia, aneuploidy, cancer, heart disease and/or sepsis.

22. The method or kit according to clauses 1 to 19 wherein the detection of the binding of the analyte is used to determine the gender of a fetus.

23. The method or kit according to clause 22 wherein the capture ligands are selected to detect a portion of the Y chromosome, preferably a portion of the SRY gene on the Y chromosome or a portion of the DYS14 or the DAZ sequence on the Y chromosome.

24. A method or kit according to any preceding clause, wherein one or more analytes are detected simultaneously.

25. A method of detecting the presence of an analyte in a sample comprising the steps of:-a) contacting the sample with a first capture ligand which is conjugated to a first metal nanoparticle whereby the first capture ligand binds to the analyte in the sample; b) contacting the first capture ligand bound to the analyte with a second different capture ligand which is conjugated to a second metal nanoparticle whereby the second capture ligand also binds to the analyte in the sample; and c) detecting the binding of the metal nanoparticles to the analyte.

26. An assay kit for use in detecting an analyte in a sample comprising:-a) a first capture ligand specific for the analyte which is conjugated to a first metal nanoparticle; and b) a second different capture ligand which is also specific for the analyte which is conjugated to a second metal nanoparticle.

27. The method of clause 25 or the assay kit of clause 26 wherein the analyte is a DNA sequence, preferably cell-free DNA, more preferably cell-free fetal DNA.

28. The method or kit according to clause 26 or 27 wherein the analyte is an RNA sequence, preferably cell-free RNA, more preferably cell-free fetal RNA.

29. The method or kit according to any of clauses 26 to 28 wherein the capture ligands are nucleic acid probes.

30. The method or kit according to clause 29 wherein the first capture nucleic acid probe is an anti-sense nucleic acid sequence and the second capture nucleic acid probe is a sense nucleic acid sequence.

31. The method or kit according to any of clauses 25 to 30 wherein the metal nanoparticic is a gold nanoparticle.

32. The method according to any of clauses 25 to 31 wherein a control capture ligand, typically an anti-sense nucleic acid sequence, is used to capture first capture ligands which have not bound to the analyte.

33. The kit according to clause 26 comprising a control capture ligand, typically an anti-sense nucleic acid sequence.

34. The method or kit according to any of clauses 29 to 33 wherein the nucleic acid probes are comprised of locked nucleic acids.

35. The method or kit according to any of clauses 25 to 34 wherein the quantitative amount of an analyte is determined.

36. The method or kit according to any of clauses 25 to 35 wherein the detection of the binding of the analyte is used to diagnose any genetic or pathological conditions typified by the presence of a specific DNA sequence.

37. The method or kit according to any of clauses 25 to 36 wherein the detection of the binding of the analyte is used to determine paternity or blood group type or is used to diagnose haemophilia, Duchenne muscular dystrophy, congenital adrenal hyperplasia, ancuploidy, cancer, heart disease and/or sepsis.

38. The method or kit according to clauses 25 to 37 wherein the detection of the binding of the analyte is used to detennine the gender of a fetus.

39. The method or kit according to any of clauses 25 to 38 wherein the capture ligands are selected to detect a portion of the Y chromosome, preferably a portion of the SRY gene on the Y chromosome or a portion of the DYS14 or the DAZ sequence on the Y chromosome.

40. A method or kit according to any of clauses 25 to 39 wherein one or more analytcs are detected simultaneously.

41. An apparatus for processing a biological sample, comprising:-a housing, the first surface of which comprises a well which extends into the housing, a hinged lid connected to the first surface of the housing, the lid comprising an outer and an inner surface, the inner surface forming a protrusion which extends into the well when the hinged lid is urged towards the first surface of the housing, the boundaries of the well being formed by the inner surface of the lid and the outer surface of the housing, a sharp within the well, a tubular member comprising a proximal and a distal end, being connected to a resilient member, said resilient member being fixed to the first surface of the housing in the well, an aperture in the first surface of the housing in the well, a membrane which can be accessed through the aperture in the first surface of the housing in the well, whereby urging of the inner surface of the lid towards the first surface of the housing causes the protrusion to enter the well and contact the tubular member, thereby compressing the resilient member and forcing the tubular member towards the membrane, and whereby the distal end of the tubular member contacts the membrane when the lid closes the well.

42. The apparatus of clause 41 additionally comprising a diagnostic test strip in fluid communication with the membrane.

43. The apparatus of clause 41 or 42, wherein the lid is prevented from being urged away from the first surface of the housing once the tubular member has contacted the membrane.

44. The apparatus of any of clauses 41 to 43 additionally comprising a buffer chamber, preferably wherein the buffer is releasable.

45. The apparatus of any of clauses 41 to 44 wherein the biological sample is blood 46. The apparatus of any of clauses 41 to 45 wherein the collection vessel is a capillary tube.

47. The apparatus of any of clauses 41 to 46 wherein the membrane is a plasma separation membrane.

48. A computer implemented method for providing information relating to a diagnosis, comprising the steps of capturing an image of an assay result obtained by the method of clauses 1 or 25 or from a kit according to clauses 2 and 26 using a camera comparing the image to existing images on a database to determine a diagnostic state transmitting information conveying the diagnostic state to a user; and providing a user selectable link to initiate communication with a remote device.

49. The computer implemented method of clause 48 wherein the remote device is operated by a healthcare professional, a counsellor or another user who has received information conveying a diagnostic state.

50. The computer implemented method of clause 48 or 49 wherein the diagnostic state is the gender of a fetus.

51. The computer implemented method of any of clauses 48 to 50 wherein the camera is a smartphone camera or a personal digital assistant camera.

52. The computer implemented method of any of clauses 48 to 51 comprising the step of storing the captured image on a database.

53. A computer readable medium storing instructions which when performed carry out the method of any of clauses 48 to 52.

54. An electronic device having a processor and a memory, the memory storing instructions which when carried out cause the processor to carry out the steps of capturing an image of an assay result obtained by the method of clauses 1 or 25 or from a kit according to clauses 2 and 26 using a camera comparing the image to existing images on a database to determine a diagnostic state transmitting information conveying the diagnostic state to the user; and providing a user selectable link to initiate communication with a remote device.

55. A method according to clauses 1 or 25, a kit according to clauses 2 or 26, an apparatus according to clause 41, a computer implemented method according to clause 49 or an electronic device having a processor and a memory according to clause 54 substantially as described herein with reference to the accompanying figures.

Claims

CLAIMS1. A method of detecting the presence of an analyte in a sample comprising the steps of:-a) contacting the sample with a first capture ligand which is conjugated to a first metal nanoparticle whereby the first capture ligand binds to the analyte in the sample; b) contacting the first capture ligand bound to the analyte with a second different capture ligand which is conjugated to a second metal nanoparticle whereby the second capture ligand also binds to the analyte in the sample; and c) detecting the binding of the metal nanoparticles to the analyte.
2. An assay kit for use in detecting an analyte in a sample comprising:-a) a first capture ligand specific for the analyte which is conjugated to a first metal nanoparticle; and b) a second different capture ligand which is also specific for the analyte which is conjugated to a second metal nanoparticle.
3. The method of claim 1 or the assay kit of claim 2 wherein the analyte is a DNA sequence, preferably cell-free DNA, more preferably cell-free fetal DNA
4. The method or kit according to claim 1 or 2 wherein the analyte is an RNA sequence, preferably cell-free RNA, more preferably cell-free fetal RNA.
5. The method or kit according to any of claims 1 to 4 wherein the capture ligands are nucleic acid probes.
6. The method or kit according to claim 5wherein the first capture nucleic acid probe is an anti-sense nucleic acid sequence and the second capture nucleic acid probe is a sense nucleic acid sequence.
7. The method or kit according to any of claims 1 to 6 wherein the metal nanoparticle is a gold nanoparticle.
8. The method according to any of claims 1 to 6 wherein a control capture ligand, typically an anti-sense nucleic acid sequence, is used to capture first capture ligands which have not bound to the analyte.
9. The kit according to claim 8 comprising a control capture ligand, typically an anti-sense nucleic acid sequence.
10. The method or kit according to any of claims 5 to 9 wherein the nucleic acid probes are comprised of locked nucleic acids.
11. The method or kit according to any of claims 1 to 10 wherein the quantitative amount of an analyte is determined.
12. The method or kit according to any of claims 1 to 11 wherein the detection of the binding of the analyte is used to diagnose any genetic or pathological conditions typified by the presence of a specific DNA sequence.
13. The method or kit according to any of claims 1 to 12 wherein the detection of the binding of the analyte is used to determine paternity or blood group type or is used to diagnose haemophilia. Duchenne muscular dystrophy, congenital adrenal hyperplasia, aneuploidy, cancer, heart disease and/or sepsis.
14. The method or kit according to claims 1 to 13 wherein the detection of the binding of the analyte is used to determine the gender of a fetus.
15. The method or kit according to any of claims I to 14 wherein the capture ligands are selected to detect a portion of the Y chromosome, preferably a portion of the SRY gene on the Y chromosome or a portion of the DYS14 or the DAZ sequence on the Y chromosome.
16. A method or kit according to any of claims 1 to 15 wherein one or more analytes are detected simultaneously.
17. A computer implemented method for providing information relating to a diagnosis by the method of claim I. comprising the steps of capturing an image of an assay result obtained by the method of claim 1 or from a kit according to claim 2 using a camera comparing the image to existing images on a database to determine a diagnostic state transmitting information conveying the diagnostic state to a user; and providing a user selectable link to initiate communication with a remote device.
18. The computer implemented method of claim 17 wherein the remote device is operated by a healthcare professional, a counsellor or another user who has received information conveying a diagnostic state.
19. The computer implemented method of claim 17 or 18 wherein the diagnostic state is the gender of a fetus.
20. The computer implemented method of any of claims 17 to 19 wherein the camera is a smartphone camera or a personal digital assistant camera.
21 The computer implemented method of any of claims 17 to 20 comprising the step of storing the captured image on a database.
22. A computer readable medium storing instructions which when performed carry out the method of any of claims 19-21.
23. An electronic device having a processor and a memory when used in the method of claim I, the memory storing instructions which when carried out cause the processor to carry out the steps of capturing an image of an assay result obtained by the method of claim 1 or from a kit according to claim 2 using a camera comparing the image to existing images on a database to determine a diagnostic state transmitting information conveying the diagnostic state to the user; and providing a user selectable link to initiate communication with a remote device.