WO2015005872A1 - Dispositif de détection d'analytes - Google Patents

Dispositif de détection d'analytes Download PDF

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Publication number
WO2015005872A1
WO2015005872A1 PCT/SG2014/000328 SG2014000328W WO2015005872A1 WO 2015005872 A1 WO2015005872 A1 WO 2015005872A1 SG 2014000328 W SG2014000328 W SG 2014000328W WO 2015005872 A1 WO2015005872 A1 WO 2015005872A1
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WO
WIPO (PCT)
Prior art keywords
sample
tubes
capillary tubes
coated
receptors
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PCT/SG2014/000328
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English (en)
Inventor
Xiaoqun Zhou
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Agency For Science, Technology And Research
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Publication of WO2015005872A1 publication Critical patent/WO2015005872A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N2021/6482—Sample cells, cuvettes

Definitions

  • the present invention generally relates to a device capable of detecting the presence or absence of one or more distinct analytes within a sample, and particularly a disposable test kit comprising the device as defined above.
  • the present invention also relates to the use of such a device for detecting the presence or absence of one or more distinct analytes in a sample.
  • the substance to be detected may be any analyte comprising a biological or chemical substance.
  • a biological analyte may comprise proteins, genes, DNA sequences and nucleotides, antigens, antibodies or any other biological substance that may be detected and identified.
  • a chemical analyte may comprise any compounds or molecules, whether organic or inorganic, or any other chemical substance that may be detected and identified.
  • Micro-chip assays typically comprise one or more sensor chips affixed on a glass, silicon or plastic substrate of several centimeters square formed with a minute flow path having a cross-sectional width of several micrometers to several millimeters by which the chemical assay procedures may be integrated.
  • receptors used for detecting and identifying a particular analyte may be coated.
  • the sample may need to be pumped through a microfluidic tube which forms the minute flow path that is in fluid communication with the micro-chip.
  • the receptors may interact with the analyte that it is capable of detecting from the sample.
  • the analyte of interest may bind to the receptor immobilized on the micro-chip and produce a chemical reaction resulting in the emission of fluorescence.
  • fluorescence is detected, identification of the particular analyte becomes possible.
  • microfluidic tube has to be washed thoroughly several times in order to prevent any contamination as it may be too troublesome or not cost effective to simply dispose and replace the microfluidic tube.
  • the abovementioned micro-chip detection assay may only allow a single sensor chip for a single analyte detection. This means that one micro-chip may only be suited to detect one type of analyte. If two different assay reactions were combined into the same micro-chip, interference may occur e.g. the colorimetric density of one reaction in some instances may overlap with the wavelengths for detection of another analyte, thus masking out the other. In another instance, the receptor necessary for the detection of a particular analyte could be detrimental to another. Therefore, in most cases, it may not be possible to use such micro-chip assays for detecting multiple types of analytes within a sample. Even if a single dye capable of emitting different wavelengths may be used, the emission of the different wavelengths may be susceptible to cross- talk when detecting different analytes concurrently thereby leading to inaccurate detection results.
  • each of these detection elements may utilize a distinct dye that emits a different wavelength from another dye present in another detection element in order to differentiate the different analytes detected so as to address the cross-talk issue or overlapping wavelengths.
  • quantum dot assemblies consisting of many different sizes of quantum dots, such as a gradient multi-layer nanofilm, may be made to exhibit a range of desirable emission properties. Since such assemblies may emit different wavelengths, each of these wavelengths may be associated with the detection of a particular analyte.
  • a device capable of detecting the presence or absence of one or more distinct analytes within a sample, said device comprising one or more multi-coated capillary tubes, each capillary tube having an opening at each opposing end, and both ends optionally connected to one end of another capillary tube to form a channel for the sample to flow through, wherein each of the one or more multi-coated capillary tubes are capable of detecting at least one of the one or more distinct analytes.
  • the one or more multi-coated capillary tubes may be connected serially.
  • such a device allows multiple analytes to be detected using a single wavelength dye. Auto-sampling can be achieved by using the capillary action provided by the capillary tubes. This overcomes the need for any pumping means.
  • the device as defined above also features one or more multi-coated capillary tubes having one or both ends connected to one end of another capillary tube to form a plurality of connected tubes, wherein the device further comprises a first opening at one end of the connected tubes for accessing said sample and an absorbent pad positioned adjacent to a second opening located at the opposing end of the connected tubes. The second opening may be in fluid communication with the absorbent pad.
  • This pad serves to collect liquid waste and can be easily disposed, thereby mitigating the need for any additional liquid waste collection chamber or the inconvenience of handling the disposal of liquid waste.
  • the plurality of multi-coated capillary tubes may be connected via silica tubes.
  • these silica tubes prevent leakage of any sample flowing through the connected multi-coated capillary tubes.
  • the multi-coated capillary tubes may be comprised of transparent glass or plastic.
  • the multi-coated capillary may be transparent or translucent.
  • each of the one or more distinct analytes that may be captured in the multi-coated capillary tubes may be indirectly bonded to a dye that emits light having a single wavelength instead of multiple wavelengths.
  • Each of the one or more distinct analytes may be bonded to a dye-conjugated secondary receptor, wherein the dye is a single wavelength dye. Use of this single wavelength dye eliminates any interference or cross-talk issues as discussed in the present disclosure.
  • the dye may be directly conjugated or indirectly conjugated to the analyte.
  • This dye may require an excitation light source in order to be activated for light emission, particularly fluorescence emission.
  • This dye may also emit light without the need for any excitation light source.
  • the one or more distinct analytes may be selected from the group consisting of antibodies, antigens, bacteria, viruses, proteins, carbohydrates, lipids, nucleic acids, organic molecules and inorganic molecules.
  • the multi-coated capillary tubes of the device as defined above may comprise an internal layer of nanoparticles. These nanoparticles may be internally coated on the capillary tubes. These nanoparticles may be zinc oxide nanorods. Advantageously, these nanoparticles or nanorods serve to increase the surface area for immobilizing receptors used for capturing the analytes to be detected.
  • the multi-coated capillary tubes may further comprise an internal layer of receptors immobilized on the nanoparticles.
  • the receptors immobilized in one multi-coated capillary tube may optionally be the same or different as the receptors immobilized in another multi-coated capillary tube and each of the immobilized receptors specifically binds to one analyte from the one or more distinct analytes in the sample flowing through the capillary channel from the first opening at one end of the plurality of connected tubes, thereby detecting the presence or absence of one or more distinct analytes within the sample.
  • the device as defined above advantageously allows detection of multiple analytes using a single wavelength dye through the use of multiple capillary tubes which may specifically detect distinct analytes.
  • the immobilized receptors as mentioned above may be selected from the group consisting of antigens, antibodies, antigen-binding fragments, enzymes, hormone-binding proteins, nucleic acid binding proteins and any other molecules capable of specifically binding to one of the one or more distinct analytes.
  • One or more of the distinct analytes in the sample flowing through the capillary channel may become bonded to these receptors that may be immobilized on the nanoparticles internally coated on said one or more capillary tubes.
  • the receptors immobilized on the capillary tubes are specific, it is possible that one or more distinct analytes in the sample may flow through the channel without bonding to any of the receptors and end up being collected on the absorbent pad of said device.
  • the device as defined above may further comprise a built in or separate excitation light source for activating the conjugated dye.
  • the activated conjugated dye may emit fluorescence of a single wavelength upon activation by to the excitation light source.
  • the device as defined above may further comprise an outer cassette which envelopes the plurality of connected multi-coated capillary tubes.
  • This cassette may comprise one or more transparent windows which allow the detection of light, particularly fluorescence, emitted by the dye. At least one of the one or more transparent windows of the device as defined above may be in an aligned position with a sensor and the excitation light source for detecting the single wavelength fluorescence emitted by the conjugated dye.
  • This sensor may be a built in sensor or a sensor existing as a separate component from the device. This sensor may be capable of detecting light of a single wavelength.
  • This sensor may be an avalanche photodiode (APD).
  • APD avalanche photodiode
  • a disposable test kit comprising a device as defined above for detecting the presence or absence of one or more distinct analytes in a sample.
  • a test kit can be easily disposed and eliminates any potential cross-contamination that may occur during the reuse of non-disposable microfluidic analyte detection assays if they are not washed thoroughly.
  • FIG. 1 depicts a top planar interior view of a disposable multi-analyte device showing the silica tubes connecting each multi-coated capillary tube in accordance with a present embodiment. The sample at one end of the connected tubes and an absorbent pad in fluid communication with the opposing end of the connected tubes in accordance with the present embodiment is also depicted.
  • FIG. 2a depicts a front-top-right perspective view of a multi-coated capillary tube in accordance with the present embodiment showing the internally coated nanoparticle layer and the innermost layer comprising the immobilized receptors.
  • FIG. 2b depicts a scanning electron microscope (SEM) image showing an open end of a capillary tube internally coated with zinc oxide nanorods in accordance with the present embodiment.
  • FIG. 2c depicts a SEM image of a capillary tube in accordance with the present embodiment showing the curvature of the optically transparent capillary tube wall and a layer of internally coated nanorods.
  • FIG. 2d depicts a magnified SEM image showing the zinc oxide nanorods internally coated on the capillary tube according to the present embodiment.
  • FIG. 2e depicts a magnified SEM image showing a bovine serum albumin (BSA) layer internally coated on the capillary tube of the present embodiment where there are no zinc oxide nanorods.
  • BSA bovine serum albumin
  • FIG. 3 a depicts a schematic diagram indicating how the zinc oxide nanorods are coated onto the inner capillary wall according to the present embodiment and subsequent silanization.
  • FIG. 3b depicts a schematic diagram showing how antibodies receptors are immobilized onto the zinc oxide nanorods and blocking of exposed surfaces of the zinc oxide nanorods.
  • FIG. 3c depicts a schematic diagram on how the immobilized antibodies receptors capture the specific analytes, the remaining recognition receptors (antibodies) and the dye- conjugated secondary receptors (antibodies). This also depicts how the detection mechanism works.
  • FIG. 4 depicts a front view of a detection sensor positioned adjacent to the outer cassette of the disposable multi-analyte device as well as the sample depicted in FIG. 1 in accordance with the present embodiment.
  • the transparent windows on the outer cassette are shown, revealing the connected capillary tubes.
  • FIG. 5 shows the detection results of different analytes within a sample at various concentrations using the device of the present embodiment.
  • the sample 112 may be a liquid comprising an organic solution or an aqueous solution.
  • the sample 112 may also comprise body fluids such as blood, plasma, serum, saliva, cerebrospinal fluid, amniotic fluid, urine, semen, effluent, soil extracts and the like.
  • analyte it may comprise any biological or chemical substance.
  • a biological or chemical substance may include antibodies, antigens, bacteria, viruses, proteins, carbohydrates, lipids, nucleic acids, DNA sequences, organic molecules or inorganic molecules.
  • the present device 100 comprises one or more multi-coated capillary tubes for detecting the presence or absence of one or more distinct analytes within a sample 112.
  • the present device 100 comprises one or more multi-coated capillary tubes 104.
  • Each of these capillary tubes 104 comprises an opening 102 at each opposing ends of the capillary tube 104. Either one of these ends may be use to contact the sample 112.
  • either one of the ends of the multi-coated capillary tubes 104 may be used to connect to the end of another capillary tube 104.
  • both ends of the capillary tube 104 may be used to connect to another capillary tube 104 to form a channel 206 (see FIG. 2a and FIG. 2b) for the sample 112 to flow through.
  • These capillary tubes may be serially connected together.
  • the one or more multi-coated capillary tubes 104 are capable of detecting the one or more distinct analytes.
  • a channel 206 is formed.
  • These connected capillary tubes 104 comprise a first opening 102 at one end of the connected tubes for accessing a sample 112.
  • a sample 112 comprising one or more distinct analytes, particularly a liquid sample 1 12
  • the liquid sample 112 is automatically drawn into the first capillary tube 104 via the opening 102 of the connected capillary tubes 104.
  • the above phenomenon occurs because of intermolecular forces between the liquid and the surrounding solid surfaces of the capillary tube 104, especially when the inner diameter of the capillary tube is sufficiently small.
  • the inner diameter of this capillary tube is at least 0.8 mm.
  • the inner diameter of this capillary tube may also be in the range of 0.1 mm to 1 mm. Particularly, the inner diameter of this capillary tube may be 0.8 mm.
  • the capillary action is due to the pressure of cohesion and adhesion which cause the liquid to work against gravity.
  • the use of a pump or the manual preparation of the sample to be pumped for passing the liquid sample 112 through the microfluidic tubes having a minute flow path is advantageously no longer required.
  • the present device 100 comprising the one or more capillary tubes 104 connected together is capable of auto-sampling via capillary suction without the need for any pumps.
  • This absorbent pad 106 may be made up of any material that is capable of absorbing any liquid sample. Such absorbent material may include cotton, wool, or any other material suitable for absorbing a liquid. This absorbent pad 106 serves to collect any liquid sample 112 that flows through the connected capillary tubes 104 by capillary action. Without this absorbent pad 106, the liquid sample that successfully passes through the connected capillary tubes may leak out of the device.
  • the amount of absorbent material used may be any amount sufficient to absorb at least 1 ml of liquid sample without any leakage.
  • the amount of absorbent material used may also be any amount sufficient to ensure that no collected liquid sample leaks out of the present device. Any skilled person may determine the amount of absorbent material used based on the dimensional configurations of the device 100 and the amount of liquid sample 112 to be used for detection. Accordingly, this absorbent pad 106 advantageously overcomes the inconvenience of using another chamber just for the purpose of collecting liquid sample waste. This absorbent pad 106 also mitigates the need for handling liquid disposal waste as it is capable of collecting the liquid sample 112 that passes through the connected capillary tubes 104. Thus, it will be easier to dispose of the liquid waste when they are consolidated and collected on the absorbent pad 106.
  • the multi-coated capillary tubes 104 are connected by silica tubes 108. These silica tubes 108 prevent the liquid sample 1 12 from leaking out of the ends of each connected capillary tubes 104. These silica tubes secure the various capillary tubes together to ensure that there is a continuous capillary action exerted on the liquid sample 112 even when it flows from one capillary tube 104 to another adjoined capillary tube 104.
  • the silica tube 108 may be opaque which means that no light may be transmitted through it.
  • the silica tube 108 may also be translucent or transparent. This means that it may allow some light or all light to pass through, respectively.
  • the multi-coated capillary tubes 104 may be made up of transparent glass or plastic.
  • the transparency of the multi-coated capillary tubes 104 is such that it allows sufficient light to pass through so that the detection of any analytes is not compromised.
  • the glass or plastic may be rigid, meaning that these materials do not flex easily and capillary tubes 104 made of such rigid materials are able to form a sufficiently straight channel 206 for the liquid sample 112 to flow through without causing any obstruction.
  • the glass or plastic may be flexible, meaning that these materials may be able to bend sufficiently without breaking and capillary tubes 104 made of such flexible materials are able to form a continuous flow channel for the liquid sample 112 to pass through with causing any obstruction even when the tubes 104 are flexed.
  • the optically transparent curved wall 208 of the capillary tubes 104 helps to facilitate the detection of the light or fluorescence emitted by a dye that may be directly or indirectly conjugated on the analyte which is in turn bounded to an immobilized receptor.
  • the curvature of the capillary wall 208 is capable of acting like a convex lens which helps to focus light emitted by the dye, thereby facilitating detection and enhancing detection sensitivity.
  • the present device 100 is capable of detecting the presence of one or more distinct analytes.
  • These one or more distinct analytes may be selected from the group consisting of antibodies, antigens, bacteria, viruses, proteins, carbohydrates, lipids, nucleic acids, organic molecules and inorganic molecules.
  • the above one or more distinct analytes may be mixed with a dye that emits light before or during the contacting of the device 100 with the sample 112 without the need for any external excitation source. During the mixing, the one or more analytes may become directly or indirectly conjugated or bonded to the light emitting dye. In another instance, the above one or more distinct analytes may be mixed with a dye that emits light only after it has become excited, which means that the dye needs to be exposed to an external light source capable of causing excitation in order for the dye to emit its own light.
  • the sample 112 containing the one or more distinct analytes, whether directly or indirectly conjugated with the dye may or may not emit light before being drawn into the present device 100 as defined above. Accordingly, such a sample may or may not be a fluorescent sample before contacting the device 100.
  • the dye may be a single or multiple wavelengths dye.
  • An example of a single wavelength dye may be a cyanine dye, such as Cy3, Cy5 etc.
  • a skilled person may also choose not to mix the sample 112 with the dye before contacting the sample 112 with the device 100 in order to avoid contamination. To do so, the skilled person may contact the device 100 with the raw sample 112 first. After sufficient liquid sample 112 has passed through all connected multi-coated capillary tubes 104, recognition receptors, in the form of a liquid solution, may be used for specifically binding to one of the one or more distinct analytes that may have bonded to the receptors immobilized on the coated capillary tubes.
  • recognition receptors may be selected from the group consisting of antigens, antibodies, antigen-binding fragments, enzymes, hormone-binding proteins, nucleic acid binding proteins and any other molecules capable of specifically binding to one of the one or more distinct analytes that have bonded with the immobilized receptors.
  • secondary receptors which specifically bind to the recognition receptors may be used. This secondary receptor may be chemically conjugated to a dye. In this manner, when the secondary receptor becomes bonded to the recognition receptor which may in turn be bonded to one of the one or more distinct anlaytes that may have been captured by the immobilized receptors, the analyte becomes indirectly conjugated with the dye without having the sample 112 contaminated with the dye in the first place.
  • secondary receptors may be selected from the group consisting of antigens, antibodies, antigen-binding fragments, enzymes, hormone-binding proteins, nucleic acid binding proteins and any other molecules capable of specifically binding to one of the one or more distinct recognition receptors.
  • FIG. 2a depicts one of the multi-coated capillary tubes 104. It can be observed that on the internal surface of the capillary wall 208, a layer of nanoparticles 202 may be internally coated. These coated nanoparticles 202 may comprise any materials made up of metals or metal oxides. These nanoparticles 202 may also exist in the form of nano-spheres, nano-cylinders, nanorods or any other suitable configuration that allows it to be coated onto the capillary wall 208. Particularly, these nanoparticles 202 may be zinc oxide nanorods.
  • the advantage of using these nanoparticles, particularly these zinc oxide nanorods, is to increase the surface area available for contact with the liquid sample 112, particularly the one or more distinct analytes contained in the sample 1 12 that flows through the channel 206. With an increased surface area, more receptors for capturing the analytes may be immobilized on these nanorods thereby increasing the receptors density. With increased receptors density, even a low concentration of analytes in a sample 112 can be detected.
  • the concentration of analytes in a sample that can possibly be detected may be at least 1 mg/ml.
  • the concentration of analytes in a sample that can possibly be detected may also be at least 10 ng/ml.
  • the intensity of the light emitted may also be increased due to a higher amount of immobilized receptors available for capturing more analytes. Accordingly, improved sensitivity is attained through the use of these nanoparticles, particularly the zinc oxide nanorods.
  • This coated layer of nanoparticles 202, particularly zinc oxide nanorods does not impede light transmission out of or into the capillary tubes 104. Hence, when nanoparticles, particularly the zinc oxide nanorods are coated on the capillary wall 208, detection of analytes is not compromised.
  • the layer 204 represents the receptors or receptor layer used for detecting the analytes.
  • the receptors comprising the receptor layer 204 is immobilized on the nanoparticles 202 and each receptor layer 204 in each distinct capillary tubes 104 may be the same or different. This means that when the multi-coated capillary tubes 104 are connected together, one or more of these tubes 104 may contain the same immobilized receptor layer 204. On the other hand, it may also mean that none of these connected multi-coated capillary tubes 104 contains the same receptor layer 204.
  • this immobilized receptor layer 204 may comprise receptors selected from the group consisting of antigens, antibodies, antigen-binding fragments, enzymes, hormone- binding proteins, nucleic acid binding proteins and any other molecules capable of specifically binding to one of the one or more distinct analytes.
  • receptors may comprise monoclonal antibodies for detecting carcinoembryonic antigen (CEA), human epidermal growth factor receptor 2 (HER2), alpha-fetoprotein (AFP) or prostate-specific antigen (PSA).
  • CEA carcinoembryonic antigen
  • HER2 human epidermal growth factor receptor 2
  • AFP alpha-fetoprotein
  • PSA prostate-specific antigen
  • FIG. 2b SEM images of the zinc oxide nanorods coated capillary tubes are shown in FIG. 2b to FIG. 2d.
  • the schematic of how these zinc oxide nanorods are coated onto the inner wall of the capillary tubes are described in the examples below and in FIG. 3a.
  • the wall of the capillary tube 208 may be first washed with alcohol or deionized water for cleaning and/or sterilization. Any other fluids with cleaning or sterilization effect may also be used. An ultrasonic cleaner may be used to aid the cleaning and/or sterilization. Subsequently, the inner surface of the wall of the capillary tube 208 may be activated for coating by contacting or soaking the whole capillary tube 104 in freshly prepared potassium permanganate solution for 0.5 h. After rinsing with deionized water, the capillary tubes 104 may be soaked completely in a solution containing the materials needed for the nanorods 301 to grow on the capillary inner wall surface.
  • the capillary tubes 104 may be soaked in a solution containing zinc nitrate hexahydrate, ammonium hydroxide and ethanolamine at a temperature of 75 C and maintained for 1.5 h without agitation. The coated capillary tubes 104 may then be removed out from the solution and rinsed with water, followed by drying in gentle N 2 flow. SEM images of the successfully coated zinc oxide nanorods 301 may be observed in FIG. 2d. This part of the scheme is represented by arrow 1 in FIG. 3 a.
  • the second part of the scheme in FIG. 3a depicted by arrow 2 represents the silanization of the nanorod 301 surface.
  • Any materials capable of being silanized on the nanorod surface may be selected by a skilled person. Such a material may also be capable of immobilizing receptors on the nanorod surface. Silanization 2 is performed so as to aid the immobilization of the receptors on the nanorod surface.
  • Such materials may be bonded with the receptors via a chemical bond or other forms of interaction e.g. hydrophilic or hydrophobic interactions, Van der Waals forces of attraction or electrostatic forces. Such materials may also be entirely replaced by the receptors once the latter is immobilized on the nanorod surface.
  • GPTS (3-glycidoxypropyl) trimethoxy silane
  • the receptors 305 are immobilized 3 on the nanorods 301, there may be empty voids between the immobilized receptors 305. This is because the surface of the nanorods 301 may not be entirely immobilized with receptors 305. These exposed surfaces may physically or chemically interact and retain any analytes as the liquid sample 1 12 flows through the multi- coated capillary tubes 104. Hence, this may cause an inaccurate detection of analytes since more than one type of analyte may be captured and retained unintentionally by such exposed nanorod 301 surface.
  • capillary tubes 104 already coated with nanorods 301 and immobilized with receptors 305 may be further coated with a substances that do not bind to any of the analytes in the sample.
  • This coating method 4 may be achieved by coating the exposed surfaces of zinc oxide nanorods 301 with a blocking substance 307.
  • the already coated capillary tube 104 may be incubated with a solution containing the blocking substance 307 e.g. albumin bovine serum (BSA).
  • the BSA particles 307 may be immobilized on the exposed surfaces of the zinc oxide nanorods 301 via any chemical bonding or other forms of physical interactions. Once the empty voids are blocked by the BSA 307, non-specific binding of analytes may be eliminated.
  • the analyte to be detected is one that may not interact with the exposed zinc oxide surface or may not be easily retained or captured by the exposed zinc oxide nanorods, the coating of a blocking substance may not be necessary.
  • FIG. 2e is a SEM image showing BSA particles blocking exposed surfaces of the zinc oxide nanorods not occupied by immobilized receptors.
  • the present device 100 is capable of detecting one or more distinct analytes as each of the receptors specifically bind to one analyte from the one or more distinct analytes in the sample 112 flowing through the channel 206.
  • each capillary tubes 104 can be configured or designed to detect a particular type of analyte within the liquid sample 112 that is able to interact with its complementary receptor. Based on this, the present device 100 is able to attain the advantage of detecting multi-analytes within a sample 112.
  • FIG. 3c shows how a specific immobilized antibody receptor 305 captures a specific complementary analyte 309 e.g. an antigen, and the subsequent dye conjugation represented by arrows 6 and 7 ⁇ Observably, when the device 100 is contacted with the liquid sample 112 and capillary action 5 forces the liquid sample through the channel 206 of the first connected capillary tube 104, one or more of the distinct analytes 309 may come into contact with the antibody receptors 305 already immobilized on the zinc oxide 301 coated capillary tubes. As the immobilized antibody receptor 305 is specific, it may only capture and bind to a complementary analyte 309 that is specific to it.
  • a specific complementary analyte 309 e.g. an antigen
  • the device may be contacted 6 with another liquid solution comprising one or more distinct recognition receptors 311.
  • These recognition receptors 311 may be specific antibodies in the sense that they may only bind to the analyte antigen 309 that is already bonded to the immobilized antibodies 305.
  • the device 100 may be contacted 7 with another solution containing the dye 315.
  • This dye 315 may be chemically or physically conjugated to a secondary receptor 313 by way of a chemical bond or other forms of chemical or physical interactions.
  • This secondary receptor may be an antibody.
  • These dye- conjugated secondary antibodies 313 may be passed through the channel 206 and contacted with the recognition antibodies receptors 311.
  • the secondary antibody 313 is also specific in the sense that it may only bind to its complementary recognition antibody receptor 311 and not the analyte antigen 309. Through this method, the dye 315 becomes indirectly conjugated to the analyte 309 to be detected. Since the various antibodies used are known, when the dye-conjugated antibodies emit light or fluorescence, it becomes possible to detect and identify the antigen that has been captured in the multi-coated capillary tube. In addition, since the raw sample need not be contacted with the dye, pre-contamination is avoided.
  • the first capillary tube 104 may contain a receptor layer 204 comprising receptors that are distinct from the receptors present in the receptor layers immobilized on subsequently connected multi-coated capillary tubes 104.
  • the first capillary tube 104 may be coated with a monoclonal reagent such as a particular type of antibody that binds specifically to only one type of antigen.
  • the second capillary tube 104 is immobilized with a different type of antibody that binds to another type of antigen.
  • the liquid sample 112 gets drawn into the first capillary tube 104 by capillary suction and passes through it, the respective analyte antigens that are able to bind to the antibodies immobilized on the first capillary tube gets isolated.
  • the liquid sample 112 then flows on into the second connected capillary tube 104.
  • the type of antibody present herein may be different from those immobilized in the first capillary tube 104 and hence it will isolate and bind with a different type of antigen.
  • the remaining connected antibody-coated capillary tubes 104 may bind with and isolate their respective distinct antigens.
  • any antigens in the sample 1 12 that are not isolated by the immobilized antibodies of any multi-coated capillary tubes 104 flow through the channel 206 of all connected capillary tubes 104. As these analytes (antigens) are unbounded, they end up being collected on the absorbent pad 106.
  • a new unused device 100 can be used since the capillary tubes 104 and absorbent pad 106 can be easily disposed. Alternatively, the whole device 100 can be easily disposed.
  • the type of antibody coated on the capillary tubes 104 are known, it becomes possible to detect and identify the type of antigen that can be isolated by its respective antibody- coated tube 104. Thus, different antigens can be isolated and detected by the different connected multi-coated capillary tubes 104. Due to such a design, the dye-conjugated secondary antibodies receptors 313 used for binding with the analyte antigens 309, or even any other analytes, need not be a multiple wavelength dye.
  • Dye of a single wavelength can be used to aid the detection of distinct multiple analytes and differentiation of the isolated analytes can be based on the different multi-coated capillary tubes 104 each having a receptor layer 204 comprising distinct immobilized receptors 305.
  • dye of a single wavelength interference of overlapping wavelengths caused by detection assays that require multiple dyes or dyes with multiple wavelengths can be avoided.
  • the cross-talk issue caused by using dyes with multiple wavelengths is also mitigated since there is no longer a need to use such dyes.
  • the present device comprises capillary tubes that are capable of being connected together, and that these capillary tubes may be coated with different detection antibody receptors, the advantageous need for only one single wavelength excitation light source and one receiver for detecting multiple analytes may be attained. Since the different coated capillary tubes of the present device are linked together, it becomes possible to use only one excitation light and one detector with one single wavelength to scan the connected multi- coated capillary tubes for detecting multiple analytes captured on these connected multi-coated capillary tubes individually i.e. the first tube captures the first type of analyte while the second tube captures the second type of analyte and so on.
  • the present device is capable of using only one wavelength, it is possible to identify the signal (light or fluorescence emitted) from the first tube as it is related to the first type of analyte when the first tube is scanned. Likewise, these detection and identification steps apply for subsequent tubes. This advantageously presents a cheap and simple method for detecting and identifying multi-analytes in one sample.
  • the sample may be mixed with the dye in a separate chamber. Different wavelengths are then used to detect and identify the different analytes. Therefore, there may be the need to use full band wavelength excitation light source and detectors. By using this method, the cost becomes much higher as compared to cost of detection and identification via the present device.
  • conventional devices as mentioned above may have to rely on multi-wavelength excitation light source and one or more receivers which requires more different wavelengths. When multi-wavelength excitation light source and receiver are used, the amount of coating on conventional devices may have to be increased in order to enhance detection accuracy due to the presence of different wavelengths. Hence, the present device may mitigate the need for more coatings.
  • the device 100 further comprises an outer cassette 110 which envelopes the connected multi-coated capillary tubes 104.
  • These multi-coated capillary tubes 104 may comprise any length sufficient to provide fluorescence emission. The length of these tubes may be less than 1 cm or at least 1 cm. These multi-coated capillary tubes may be 1.8 cm long.
  • the outer cassette may be of any length sufficient to house the number of capillary tubes required. This outer cassette may be less than 7 cm or at least 7 cm.
  • the outer cassette 110 may comprises one or more transparent windows 402. Each of these transparent windows 402 may be less than or more than 1 cm wide. Each of these transparent windows 402 may also be 1cm wide.
  • the capillary tube wall remains sufficiently clear to allowed light or fluorescence to pass through for detection.
  • the device may be placed in a diode sensor 404 e.g. avalanche photodiode (APD).
  • APD avalanche photodiode
  • This diode sensor 404 may comprise a light filter or a simple broadband light filter, an excitation light source, a curved lens to focus the excitation light source onto the transparent window, a holder for securing the device and a sensor that detects the fluorescence or light emitted by the excited dye. There may be another curved lens to focus the light emitted by the conjugated dye from the transparent window to the sensor. Such a device may be easily constructed based on the knowledge of a skilled person.
  • the components of the diode sensor 404 as described above may be built into the diode sensor or may exist as a separate individual component.
  • the device 100 may be placed in the holder such that the transparent window of the outer cassette is aligned with the curved lens, the excitation light source and the detection sensor.
  • the conjugated dye becomes excited and is activated to emit light, particularly fluorescence.
  • the excitation light may be any wavelength sufficient to cause the dye to emit light.
  • the excitation light may have a wavelength in the range of 400 nm to 700 nm or any other wavelength outside this range.
  • the excitation light may have a wavelength of 475 nm.
  • the excitation light source may have single or multiple wavelengths.
  • the light or fluorescence emitted by the conjugated dye may be any wavelength suitable for detection.
  • This conjugated dye may comprise any single wavelength in the range of 400 to 700 nm.
  • This conjugated dye may have any single wavelength from 550 nm to 600 nm.
  • the filter then eliminates any background light, particularly the excitation light source, allowing only the single wavelength fluorescence to pass through the filter into the detection sensor.
  • the diode sensor 404 may be capable of detecting light of a single wavelength or multiple wavelengths. Since the device 100 advantageously allows a dye capable of emitting only a single wavelength to be used, any equipment with a fixed wavelength sensing head can be used to scan the different pieces of capillary tubes 104 for detecting and identifying the distinct analytes.
  • a cheap sensor diode 404 capable of only detecting a single wavelength emitted by the dye-conjugated analytes may also be used e.g. an avalanche photodiode (APD).
  • APD avalanche photodiode
  • the other advantage of using an APD is that it may easily be incorporated into any portable device or upgraded into a portable device for convenient clinical detection of one or more distinct analytes.
  • the present device 100 as defined above is used for detecting the presence or absence of one or more distinct analytes in a sample 112 as described above.
  • a disposable test kit comprising the present device 100 as defined above available for detecting the presence or absence of one or more distinct analytes in a sample 112 as described above.
  • the exemplary embodiments are only examples, and are not intended to limit the scope, applicability, operation, or configuration of the invention in any way. Rather, the foregoing detailed description will provide those skilled in the art with a convenient road map for implementing an exemplary embodiment of the invention, it being understood that various changes may be made in the function and arrangement of elements and method of operation described in an exemplary embodiment without departing from the scope of the invention as set forth in the appended claims.
  • Capillary tubes (internal diameter 0.8 mm, from Fisher Scientific) were ultrasonically cleaned in ethanol and deionized water, followed by activating through soaking in 30mL of freshly prepared potassium permanganate (5mM) solution for 0.5 h (Fig. 3a). After intensive rinsing with deionized water, the capillary tubes were placed in a beaker (slightly inclined) with 50mL 0.1 M zinc nitrate hexahydrate solution containing 4% (v/v) of ammonium hydroxide (28 wt%) and 10% (v/v) of ethanolamine (>98%). The beaker was placed in 75°C water bath and maintained for 1.5 h without agitation.
  • 5mM potassium permanganate
  • the capillary tubes (called ZnO capillary tubes hereafter) were then removed out from the solution and rinsed with water, followed by drying in gentle N 2 flow.
  • the above methods may also be solely applied to the inner surface of the capillary tubes so that the ZnO nanorods do not get coated on the external surface of the capillary tubes. This can be observed from the SEM image in FIG. 2c.
  • ZnO capillary tubes were incubated with 5% (v/v) (3-glycidoxypropyl) trimethoxy silane (GPTS) ethanol solution for 2 h, followed by curing at 110°C in vacuum for 2 h (Fig. 3a).
  • Antibody receptors were written on the GPTS-modified ZnO by soaking the ZnO capillary tubes in a solution containing 250 ug/mL of monoclonal antibody (anti-HER2, anti-PSA and anti-AFP) in 0.01 M phosphate buffered saline (PBS) containing 2.5% glycerol and 0.004% triton X-100 was used for coating.
  • PBS phosphate buffered saline
  • the capillary tubes were blocked with 10 mg/mL bovine serum albumin (BSA) solution for 1- h to eliminate the nonspecific binding of proteins and finally washed with 0.05 M Tris buffered saline (TBS).
  • BSA bovine serum albumin
  • TBS Tris buffered saline
  • the printing buffer was optimized by varying concentrations of glycerol and surfactant in 50 ug/mL Cy3- labelled anti-goat IgG/0.01 M PBS. Alternatively, these steps may also be applied solely to inner surface of the capillary tubes.
  • the antibody coated capillary tubes were first incubated with sample solutions containing 10% human serum (in clinical blood diagnosis, 10 folds dilution) for 2 h. After rinsing with TBS, they were allowed to sequentially react with the recognition antibody receptors (rabbit anti- HER2, rabbit anti-PSA and rabbit anti-AFP in serum, 500 fold dilution) for 1 h, and also with the dye-conjugated secondary antibody receptors comprising 2 ug/mL Cy3 -labelled anti-rabbit IgG for 0.5 h (see Fig. 1). After intensive washing with TBS and deionized water, the capillary tubes were dried at 37°C before fluorescent imaging.
  • recognition antibody receptors rabbit anti- HER2, rabbit anti-PSA and rabbit anti-AFP in serum, 500 fold dilution
  • the dye-conjugated secondary antibody receptors comprising 2 ug/mL Cy3 -labelled anti-rabbit IgG for 0.5 h (see Fig. 1). After intensive washing with TBS
  • FESEM Field emission scanning electron microscope

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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Optics & Photonics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

L'invention concerne un dispositif 100 pouvant détecter la présence ou l'absence d'un ou de plusieurs analytes distincts dans un échantillon. Ce dispositif 100 comprend un ou plusieurs tubes capillaires à revêtements multiples, chaque tube capillaire présentant une ouverture en chaque extrémité opposée et les deux extrémités pouvant être reliées à une extrémité d'un autre tube capillaire pour former un canal au travers duquel l'échantillon peut s'écouler. Chacun dudit un ou desdits plusieurs tube(s) capillaire(s) à revêtements multiples peu(ven)t détecter au moins un dudit un ou desdits plusieurs analyte(s) distinct(s) qui a/ont été conjugué(s) directement ou indirectement avec un colorant qui émet une longueur d'onde unique.
PCT/SG2014/000328 2013-07-10 2014-07-10 Dispositif de détection d'analytes WO2015005872A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017188463A1 (fr) * 2016-04-25 2017-11-02 주식회사 디엠엑스 Dispositif d'analyse de fluide corporel, biocapteur, et procédé de production de biocapteur
CN110632287A (zh) * 2019-08-15 2019-12-31 成都工业学院 一种含探针阵列的毛细管及其应用和制备方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002054052A1 (fr) * 2001-01-08 2002-07-11 Leonard Fish Instruments et procedes de diagnostic permettant de detecter des analytes
US7708944B1 (en) * 2006-06-13 2010-05-04 Research Foundation Of State University Of New York Ultra-sensitive, portable capillary sensor
US20130157286A1 (en) * 2007-05-04 2013-06-20 Opko Diagnostics, Llc Fluidic connectors and microfluidic systems

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002054052A1 (fr) * 2001-01-08 2002-07-11 Leonard Fish Instruments et procedes de diagnostic permettant de detecter des analytes
US7708944B1 (en) * 2006-06-13 2010-05-04 Research Foundation Of State University Of New York Ultra-sensitive, portable capillary sensor
US20130157286A1 (en) * 2007-05-04 2013-06-20 Opko Diagnostics, Llc Fluidic connectors and microfluidic systems

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017188463A1 (fr) * 2016-04-25 2017-11-02 주식회사 디엠엑스 Dispositif d'analyse de fluide corporel, biocapteur, et procédé de production de biocapteur
CN110632287A (zh) * 2019-08-15 2019-12-31 成都工业学院 一种含探针阵列的毛细管及其应用和制备方法

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