High sensitivity fluorescent detection system

Abstract

This invention relates to a detection system for measuring a fluorescent signal in a fluorescent assay. The system comprises a gold-plated solid surface bound with a fluorescent label, a light source, a light correcting lens, and an optical detector to detect the fluorescent emission light. The solid is made of plastic doped with glass fibers and the solid surface is coated with gold, and the hardness of the solid is at least 75 based on Rockwell M hardness scale. The system provides high sensitivity and low variability for fluorescent immunoassays.

Description

[0001] HIGH SENSITIVITY FLUORESCENT DETECTION SYSTEM

[0002] FIELD OF THE INVENTION

[0003] This invention relates to a high sensitivity detection system for measuring a fluorescent signal in a fluorescent assay. The system comprises a gold-plated solid surface bound with a fluorescent label, a light source, and an optical detector to detect the fluorescent emission light. The solid is made of plastic doped with glass fibers and the solid surface is coated with gold.

[0004] BACKGROUND OF THE INVENTION

[0005] In the development of immunoassay systems, many performance requirements need be met. Assays need be sensitive enough to detect analyte at very low levels in the subpicogram nanogram range. Total assay rime needs to be 15 minutes or less in order to provide timely results for patient management in point of care situations, or to meet throughput requirements for batch analyzers. In some cases, analyte panels where multiple assays are simultaneously performed with the same sample are advantageous to minimize the turnaround time for results and test costs.

[0006] Many immunoassays employ fluorescent labels because such labels offer many practical advantages. Compared to enzymes, fluorescent labels are much more stable and do not require an additional substrate reagent. For multianalyte panels, fluorescent labels enable the use of discrete binding zones within a common reaction chamber since each binding zone can be sequentially subjected to fluorescence excitation and emission measurements without interference from adjacent binding zones. Assays utilizing fluorescent labels, however, are less sensitive than enzyme-based assays primarily due to the enzyme’s ability to catalytically convert substrate to accumulate a great amount of product molecules over time.

[0007] Ar lsulfonate cyanine fluorescent dyes are described in Mujumdar et al. (1993) Bioconjugate Chemistry, 4: 105-111; Southwick et al. (1990) Cytometry, 11:418-430; and U.S. Pat. No. 5,268,486. Cy5 is described in each of the references and is commercially available from Biological Detection Systems, Inc., Pittsburgh, Pa., under the tradename FLUOROLINK™ Cy5™. The arylsulfonate cyanine fluorescent dyes have high extinction coefficients (typically from 130,000 L / mole to 250,000 L / mole), good quantum yields, fluorescent emission spectra in a range (500 nm to 750 nm) outside of the autofluorescence

[0008] - 1 -

[0009] 183895916 1 wavelengths of most biological materials and plastics, good solubilities, and low non-specific binding characteristics.

[0010] Due to the increasing demand for fast, accurate, and early diagnosis, there is a growing demand for higher detection sensitivity. The ways to improve the sensitivity of detection include amplifying the signal and reducing the background of the detection. For the fluorescence-based detection systems, utilizing the performance of metal enhanced fluorescence (MEF) has potential to improve detection sensitivity. When the size of metallic materials is reduced from the macro-scale to the micrometer and nanometer levels, many physical and chemical properties such as light, electricity, and magnetism undergo peculiar changes. For example, when the distance between fluorescent molecules and micro / nanoscale metal materials is appropriate and the optical properties match, the fluorescence signal of fluorescent molecules can be greatly enhanced. This fluorescence enhancement phenomenon is called metal enhanced fluorescence effect, which is caused by the localized surface plasmon resonance effect and plasmon coupling effect of metals. It is mainly related to the type, shape, nanoscale of metal materials, and the distance between fluorescent molecules and metals materials, as well as the degree of matching between the localized surface plasmon absorption spectrum of metals and the excitation and emission spectra of fluorescent molecules. When the spectra of metals’ plasmon and the fluorophore overlap, the fluorescence can be enhanced [Analyst, 2015, 140. 386-406. page 394],

[0011] There is a need for an improved optical detection system and an improved method for detecting analytes with high sensitivity7by fluorescent immunoassay. The system and method should be easy to handle by the users and provide high specific signal and minimal background noise.

[0012] BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 depicts one detection system of the present invention where the solid surface is the sensing surface of a pin.

[0014] FIG. 2 illustrates one fluorescent immunoassay format.

[0015] FIG. 3 illustrates another fluorescent immunoassay format.

[0016] FIG. 4 shows linear range and sensitivity of LH immunoassay with gold-plated PPS dopped with 65% glass fiber pin.

[0017] FIG. 5. Linear range and sensitivity' of FSH immunoassay with gold-plated PPS dopped with 65% glass fiber pin

[0018] - 2 -

[0019] 183895916 1 DETAILED DESCRIPTION OF THE INVENTION

[0020] Definitions

[0021] Terms used in the claims and specification are to be construed in accordance with their usual meaning as understood by one skilled in the art except and as defined as set forth below.

[0022] "‘About / ’ as used herein, refers to within ± 10%, preferably ± 5%, of the recited value.

[0023] An "analyte-binding" molecule, as used herein, refers to any molecule capable of participating in a specific binding reaction with an analyte molecule. Examples include but are not limited to, (i) antigen molecules, for use in detecting the presence of antibodies specific against that antigen; (ii) antibody molecules, for use in detecting the presence of antigens; (iii) protein molecules, for use in detecting the presence of a binding partner for that protein; (iv) ligands, for use in detecting the presence of a binding partner; or (v) single stranded nucleic acid molecules, for detecting the presence of nucleic acid binding molecules.

[0024] An “aspect ratio” of a shape refers to the ratio of its longer dimension to its shorter dimension.

[0025] A “binding molecular,” refers to a molecule that is capable to bind another molecule of interest.

[0026] “A binding pair,” as used herein, refers to two molecules that are attracted to each other and specifically bind to each other. Examples of binding pairs include, but not limited to, an antigen and an antibody against the antigen, a ligand and its receptor, complementary strands of nucleic acids, biotin and avidin, biotin and streptavidin, lectin and carbohydrates. Preferred binding pairs are biotin and streptavidin, biotin and avidin, fluorescein and antifluorescein, digioxigenin / anti-digi oxi genin. Biotin and avidin, including biotin derivatives and avidin derivatives such as streptavidin, may be used as intermediate binding substances in assay protocols employing complex binding sequences. For example, antibodies may be labeled with biotin ("biotinylated") and used to bind to a target substance previously immobilized on a solid phase surface. Fluorescent compositions according to the present invention employing an avidin or streptavidin may then be used to introduce the fluorescent label.

[0027] "Fiberglass" is a type of fiber-reinforced plastic using glass fiber.

[0028] “Immobilized,” as used herein, refers to reagents being fixed to a solid surface. When a reagent is immobilized to a solid surface, it is either be non-covalently bound or covalently

[0029] - 3 -

[0030] 183895916 1 bound to the surface.

[0031] “A monolithic substrate / ’ as used herein, refers to a single piece of a solid material such as glass, quartz, or plastic that has one refractive index.

[0032] “An optical fiber,” as used herein, is a glass or plastic fiber that carries light along its length. An optic fiber is typically a circular cross-section dielectric waveguide consisting of a dielectric material (a core material) surrounded by another dielectric material with a lower refractive index (cladding).

[0033] A “pin,” as used herein, refers to a solid substrate in an elongated shape that has a distal end and a proximal end. The proximal end has a sensing surface and is plated with gold. The sensing surface is further coated with a thin layer of analyte-binding molecules.

[0034] Fluorescent Detection System

[0035] The present invention relates to a fluorescent detection system for measuring a fluorescent signal on a solid surface (a biosensor), where the solid surface is coated with a metal to enhance the analyte-specific fluorescence signal while reducing non-specific background noise to improve detection sensitivity. The whole solid surface or a part of the solid surface is plated with metal. The metal-plated surface is used for binding a molecule and then for detection and analysis of a biochemical event. After the metal-plated surface completes its immunoassay with an analyte and a fluorescent-labeled reagent, a fluorescent signal can be detected using an optical detection system. The excitation light excites the fluorescent labels of the immune complex bound to the metal-plated surface, while the optical detector collects the emission signal from the metal-plated surface. The present system improves the sensitivity of a detection system due to its special metal surface characteristics and optical detection methods.

[0036] In general, the fluorescent detection system of the present invention comprises: a solid surface bound with a fluorescent label; a light source for emitting excitation light to a solid surface; a light collecting lens pointed tow ard the sensing surface for collecting the emission fluorescent light; and an optical detector for detecting the emission fluorescent light; where the collecting lens collects and directs the emission fluorescent light to the optical detector; wherein the solid is made of plastic doped with glass fiber and the solid surface is plated with gold, and the hardness of the solid is at least 75 based on Rockwell M hardness scale.

[0037] The solid (the biosensor) of the present system is made of plastic doped with glass

[0038] - 4 -

[0039] 183895916 1 fiber, wherein the hardness of the solid is at least 75 based on Rockwell M hardness scale (see data from SpecialChem SA, https: / / ornnexus.specialchem.com / polymer- property / hardness-rockwell-m).

[0040] Plastic can be conveniently manufactured by injection-molding and its cost is low. However, some plastic materials do not provide sufficient hardness. The inventors have selected certain plastics doped with glass fibers and then plated with gold. The higher the doping amount of glass fiber, the greater the hardness. For an assay to work with the disclosed fluorescent detection system, the raw material of the biosensor should be able to hold the precise shape without warp. The raw material should not bend or twist out of shape during the assay or during the optical reading steps.

[0041] The inventors have selected certain plastic and dope it with glass fibers to provide sufficient hardness and collimation, which make the coordinate positioning of pin more accurate during fluorescence reading. In addition, glass fiber-doped plastics are selected for gold-plating based on the criteria that the gold-plated, glass fiber doped plastics provide high sensitivity and low variation in fluorescent immunoassays.

[0042] In one preferred embodiment, the plastic is polyphenylene sulfide (PPS), polyamide (PA), polycarbonate (PC), poly methyl methacrylate (PMMA), polystyrene (PS), or acrylonitrile butadiene styrene (ABS); and the plastic is doped with glass fiber.

[0043] In another embodiment, the plastic is polybutylene terephthalate (PBT), poly etherimide (PEI), polyethersulfone (PESU), polymethylpentene (PMP), polyphthalamide (PPA), polyphenyl sulfone (PPSU), polysulfone (PSU), styrene methyl methacrylate(SMMA). thermoplastic polyimide (TPI). polyimide (PI), polyetheretherketone (PEEK), or polyoxymethylene (POM); and the plastic is doped with glass fiber.

[0044] In yet another embodiment, the plastic is poly vinylidene fluoride (PVDF), polyethylene terephthalate (PET), liquid crystal polymer (LCP). cellulose acetate (CA), ethylene vinyl alcohol (EV OH), polyamide-imide (PAI), polyacrylonitrile (PAN), polyarylamide (PARA), or styrene-acrylonitrile copolymer (SAN); and the plastic is doped with glass fiber.

[0045] In one preferred embodiment, the solid is made of PPS doped with 30-70% glass fiber, PC doped with 20-40% glass fiber, PC doped with 20-40% glass fiber, ABS doped with 20-40% glass fiber, PA doped with 15-30% glass fiber, PARA doped with 30% -60% glass fiber, PBT doped with at least 30% glass fiber, PEEK doped with 20-40% glass fiber, PEI

[0046] - 5 -

[0047] 183895916 1 doped with 20-40% glass fiber, PPA doped with at least 45% glass fiber, PSU doped with at least 30% glass fiber. SAN doped with 20% glass fiber, or PS doped with 20-40% glass fiber.

[0048] The above-mentioned plastic doped with glass fiber provides a sufficient hardness of the solid surface, which ensures accurate positioning of the solid surface during instrument's readings. Also, the adsorption of protein by the present material is low. Solid materials with low non-specific adsorption to proteins are preferred for immunoassays. Because the sides of the pin are often not gold-plated to reduce cost, using solid materials with low non-specific adsorption to proteins reduces non-specific adsorption of proteins on the sides of pins. Further, glass fiber-doped PPS can be injection-molded, which is cheap and easy for production processing and manufacturing.

[0049] The plated metal on the solid can be any metal or alloy, such as gold, silver, copper, aluminum, chromium, titanium, and tantalum. A preferred metal is gold or silver.

[0050] The solid may be in a form of a pin, a microwell, a chip, a rod, a bead, a ball, a microfluidic channel, a reaction cup. a chromatographic a membrane, or other forms.

[0051] In one embodiment, the thickness of the gold plated on the solid surface is 50-700, or 75- 700 nm, or 100-700 nm, or 50-500 nm, or 75-500 nm, or 100-500 nm, or 50-300 nm, 75- 300 nm, or 100-300 nm. A preferred thickness is 50-500 nm. When the gold thickness is less than 50 nm, the enhancement of fluorescent signal in a fluorescent immunoassay assay is less. When the gold thickness is more than 500 nm, the increased cost of gold plating outweighs the small fluorescent signal enhancement.

[0052] In one embodiment, the solid can be any shape such as rod, cylindrical, round, square, triangle, etc., with an aspect ratio of length to width of at least 5 to 1, preferably 10 to 1. In one embodiment, the solid is a pin having an aspect ratio of length to width at least 5 to 1, or at least 10 to 1 ; the pin having a distal end and a proximal end, and the solid surface is a sensing surface at the proximal end. In one embodiment, the pin's sensing surface has a diameter equal or less than 5 mm.

[0053] The reaction of biomolecular interaction and the detection of fluorescence signal are carried out in the metal-plated part. The non-metal plated part of the solid is desirable to have low non-specific binding to proteins in the reagents during an immunoassay.

[0054] In the process of detecting a biochemical event, the solid surface carries out reactions. The process involves transferring the solid (biosensor) between sample and different reagent solutions. In order for efficient reaction and wash, fast transfer between solutions, easy signal

[0055] - 6 -

[0056] 183895916 1 detection, convenience for mass production, and a high degree of automation and highly reliability, the solid can be designed with one end having a moving mechanism holding part, and the other end (the solid surface or the sensing surface) having biochemical reactions. After the reaction is completed, the fluorescent signal of the solid surface is read by a fluorescent detector. The solid can be designed as a needle-like pin, and the sensing face of the pin can be circular, triangular, polygon, irregular shape, etc. The solid surface can be a smooth and flat plane, convex, pitted, array with pits or protrusions, or special patterns.

[0057] Different metals have different localized surface plasmon absorption spectra at different nanoscale, the enhancement of fluorescence occurs when the localized surface plasmon absorption spectra of metals overlap with the excitation or emission spectra of fluorescent molecules.

[0058] The metal plating process on the solid surface can be electrochemical coating, chemical vapor deposition (CVD), physical vapor deposition (PVD), or others. In one embodiment, vacuum magnetron ion sputtering is used for metal plating because it provides a high sputtering rate, a low substrate temperature rise, a strong adhesion between the coated materials and substrate, and a stable product. The metal-plated part can cover all or part of the solid surface. However, due to cost and manufacturing convenience, the solid surface may be coated with a metal film only on one side. For example, a pin may be only coated on its tip.

[0059] In one embodiment, a layer of metal is plated on one end face of a solid by vacuum magnetron ion sputtering. The solid should be cleaned to remove organic matters and particulate matters before metal-plating. For example, the solid is cleaned with ethanol, ultrasonic, plasma, or other methods.

[0060] Before immobilizing biomolecules on the metal-plated surface of the pin, the metal- plated-solid is also cleaned. The cleaning treatment of the solid substrate before gold plating and after gold plating are both important to provide an accurate fluorescence detection. Common surface cleaning methods of the gold-plated surface include ultrasonic cleaning, oxidation treatment, reduction treatment, oxidation-reduction treatment, organic reagent cleaning, and surfactant treatment. For example, H2O2 treatment on the gold surface is an effective and convenient cleaning method for industrial production.

[0061] In one embodiment, the steps for cleaning metal-plated solid surface include: washing the solid surface in anhydrous ethanol for a first period of time (e.g., 1-3 minutes, or 2 minutes), and then washing the solid surface with pure water. After that, washing the solid

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[0063] 183895916 1 surface in a an aqueous H2O2 solution for a second period of time (e.g., 5-15 minutes, or 10 minutes) and finally washing with pure water. The cleaned metal-plated solid surface is then ready to bind biomolecules onto the surface.

[0064] In one embodiment, pins can be assembled into a bundle for end coating.

[0065] The end or one side of the metal-plated biosensor is coated with substances that can be used for bioassay and analysis, such as proteins, antibodies, nucleic acids, antigens, and ligands, to achieve capture or reaction of the analyte, and then react with a reagent that can bring fluorescence signals. By collecting and analyzing the fluorescence signals of fluorescent-labeled substances on the biosensor, the concentration or content of the analyte can be detected.

[0066] FIG. 1 depicts one detection system of the present invention where the solid surface is the sensing surface of a pin.

[0067] Any light source that can emit proper excitation light for the fluorescent label is suitable for the present invention. A preferred light source is a laser that can emit light with wavelengths suitable for fluorescent labels. For example, the laser center wavelength is preferred to be 649 nm for Cy5 fluorescent dye. A suitable optical detector for detecting emission light is a photomultiplier tube (PMT), a charge coupled device (CCD), or a photodiode.

[0068] The light source and the optical detector including the collecting lens are mounted on the same side of the pin tip surface (the sensing surface). If the sensing surface faces down, they are both mounted below the tip surface. The sensing surface is often within the numeric aperture of the collecting lens. The pin can be. but does not have to be, centrally aligned with the collecting lens.

[0069] In FIG. 1, fluorescent labels are bound to the sensing surface. To detect fluorescence, an optical detector and an excitation laser are mounted on the same side of the pin, and the excitation light focusing lens is underneath the sensing surface. To detect the emission at the sensing surface, a photo multiplier tube or CCD can be used. The distance between the sensing surface and the emission light focusing lens is adjustable to achieve the best detection efficiency. FIG. 1 illustrates that the incident angle of the excitation light is emitted vertically to the end face of the pin. The excitation light of the laser passes through the collimator and then is reflected by the semi-reflective mirror to the converging lens. The excitation light passing through the condensing lens illuminates the metal-coated end face of the pin

[0070] - 8 -

[0071] 183895916 1 vertically. The spot area of the excitation light passing through the condensing lens is slightly smaller than the metal coating area of the pin. Since the excitation light is perpendicular to the pin end face, the size of the light spot collected on the pin end face does not change due to changes in the distance between the pin end face and the optical system. When the fluorescent molecules on the end face or the sensing surface of the pin are excited by the laser, the fluorescence passes through the condensing lens and the semi-transparent and semi- reflective mirror, passes through the filter, and then is focused by the converging lens to photodetectors such as APD, CCD, and photon counters.

[0072] When the incident angle of the excitation light is perpendicular or vertical, the laser excitation light is directly projected on the pin surface with a certain incidence after being aligned by the collimator. The fluorescence signal generated after the fluorescent molecules on the sensing surface are excited by the laser will pass through the converging lens to photodetectors such as APD, CCD, and photon counters. By adjusting the distance between the optical system and the end face of the pin, an optimal convergence of the excitation light on the pin surface and the detection of the fluorescence signal by the photoelectric detection system can be achieved. However, when the incident angle of the excitation light is not perpendicular or vertical, the coverage area of the excitation light spot is very sensitive to the distance between the optical system and the pin. A small deviation in the distance between the optical system and the pin may lead to a significant deviation in the position of the excited light spot on the pin.

[0073] In the present fluorescence detection system, the incident angle of the excitation light is preferred to be vertical or co-axial and not oblique. The co-axial excitation light reduces the coefficient of variance (CV) of an immunoassay.

[0074] Detecting an Analyte by a Fluorescent Immunoassay

[0075] The present invention is also directed to methods of detecting an analyte in a liquid sample by a fluorescent immunoassay, using a fluorescent detection system.

[0076] FIG. 2 illustrates one embodiment of the methods. In this embodiment (two-step binding), the method comprises the steps of (a) obtaining a gold-plated solid surface such as a pin having a first antibody immobilized on the tip (sensing surface) of the pin; (b) dipping the pin tip into a sample vessel containing a liquid sample having an analyte (an antigen analyte); (c) dipping the pin tip into a reagent vessel containing a reagent solution comprising

[0077] - 9 -

[0078] 183895916 1 a polymer having a molecular weight of at least 1 million Daltons and conjugated with at least 5 second antibody molecules and at least 25 fluorescent labels to form an immunocomplex of the analyte, the first antibody, and the second antibody on the pin tip; (d) dipping the pin tip into a washing vessel containing a wash solution, and (e) detecting the immunocomplex formed by detecting the fluorescent signal on the pin tip; wherein the first antibody and the second antibody are antibodies against the analyte. In the above method, an optional washing step can be added after the binding step (b).

[0079] In another embodiment, the method comprises the steps of: (a) obtaining a gold-plated pin having a first antibody immobilized on the tip of the pin; (b) dipping the pin tip into a sample vessel containing (i) a liquid sample having an analyte and (ii) a reagent solution comprising a polymer having a molecular weight of at least about 1 million Dalton and conjugated with at least 5 second antibody molecules and at least 25 fluorescent label, to form an immunocomplex of the analyte, the first antibody, and the second antibody on the pin tip; (c) dipping the pin tip into a washing vessel containing a wash solution; and (d) detecting the immunocomplex formed by detecting the fluorescent signal on the pin tip; wherein the first antibody and the second antibody are antibodies against the analyte.

[0080] In yet another embodiment (three-step binding), the method comprises the steps of: (a) obtaining a gold-plated pin having a first antibody immobilized on the tip of the pin; (b) dipping the pin tip into a sample vessel containing a sample solution having an analyte; (c) dipping the pin tip into a reagent vessel containing a reagent solution comprising a second antibody molecules conjugated with a first member of a binding pair; (d) dipping the pin tip into an amplification vessel containing an amplification solution comprising a polymer having a molecular w eight of at least about 1 million Dalton and conjugated with at least 5 molecules of second member of the binding pair and at least 25 fluorescent labels, to form an immunocomplex among the analyte, the first antibody, the second antibody, and the first and the second members of the binding pair on the pin tip; (e) dipping the pin tip into a washing vessel containing a w ash solution; and (f) detecting the immunocomplex formed by detecting the fluorescent signal on the pin tip; wherein the first antibody and the second antibody are antibodies against the analyte. In the above method, optional washing steps can be added after the binding steps (b) and (c). The extra washing steps may not be required because the amount of the carried-over solution is minimal due to a small binding surface area. In one embodiment, the first member of the binding pair is biotin, and the second member of the

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[0082] 183895916 1 binding pair is streptavidin. This Assay format is shown in FIG. 3.

[0083] In the above method, the first antibody may be attached directed on the sensing surface of the gold-plated pin. Alternatively, the first antibody may be attached to the crosslinked FICOLL® (copolymers of sucrose and epichlorohydrin) and then coated on the tip of the gold-plated pin. Cross-linked FICOLL® has low non-specific binding to proteins and reduces the background of a fluorescent assay.

[0084] In the above method, the fluorescent dye molecules may be attached directly to crosslinked FICOLL®. Alternatively, the fluorescent dye molecules are attached indirectly to the cross-linked FICOLL® through the binding molecules such as antibody molecules or streptavidins.

[0085] Methods to immobilize reagents to the solid phase (the sensing surface of the pin) are common in immunochemistry and involve formation of covalent, hydrophobic or electrostatic bonds between the solid phase and reagent. Analyte-binding molecules can be directly immobilized on the sensing surface. Alternatively, analyte-binding molecules can be indirectly immobilized on the sensing surface through a binding pair. For example, antifluorescein can be first immobilized either by adsorption to the solid surface or by covalently binding to aminopropylsilane coated on the solid surface. Then the analyte-binding molecule that is labeled with fluorescein can be bound to the solid surface through the binding of fluorescein and anti-fluorescein (binding pair).

[0086] When the binding molecule is a polypeptide or protein, such as an antibody, the fluorescent label can covalently bind to it through a variety of moieties, including disulfide, hydroxyphenyl, amino, carboxyl, indole, or other functional groups, using conventional conjugation chemistry as described in the scientific and patent literature. Alternatively, antibodies can be biotinylated by known techniques (see Wilchek and Bayer, (1988) ANAL. BIOCHEM. 171 : 1-32) and linked to the fluorescent label via avidin / streptavidin molecules.

[0087] Covalent binding of the fluorescent label to a polynucleotide can be achieved through a variety of moieties, including aldehyde, ketone, isothiocyanate, imidate, inosine, acyl, and alkyl, using conventional conjugation chemistry, while derivatization with biotin is taught in many references. (Leary et al. (1983) Proc. Natl. Acad. Sci. USA 80:4045-4049;

[0088] WO86 / 02929; EP063 879; Langer et al. (1981) Proc. Natl. Acad. Sci. USA 78:6633-6637; and EP2009 996).

[0089] Exemplary' techniques for binding arylsulfonate cyanine fluorescent dye labels to

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[0091] 183895916 1 antibodies and other proteins are described in U.S. Pat. Nos. 5,268,486; 5,650,334; the contents of which are in incorporated herein by reference. Techniques for linking a preferred Cy5 fluorescent label to both antibodies and nucleic acids are described in a technical bulletin identified as Cat. No. A25000, published by Biological Detection Systems, Inc., Pittsburgh, Pa.

[0092] The methods of the present invention can be detected by the fluorescent detection systems as described above in this application.

[0093] The invention is illustrated further by the following examples that are not to be constmed as limiting the invention in scope to the specific procedures described in them.

[0094] EXAMPLES

[0095] Materials

[0096] Crosslinked FICOLL® 400. Cy 5-labeled crosslinked FICOLL®. anti-fluorescein isothiocyanate (FITC) antibody- crosslinked FICOLL®, Cy5-streptavidin-crosslinked FICOLL®, were prepared according to the protocols described in U.S. Patent No. 10,379,116.

[0097] Example 1. Preparing gold-plated pins

[0098] Quartz, glass, and acrylic rod pins were provided by the suppliers.

[0099] Cylindrical rods pin made of different materials such as polypropylene (PP), polyamide (PA), 40% glass fiber doped PA (PA40GF), polyphenylene sulfide (PPS), 40% glass fiber doped PPS (PPS40GF). and 65% glass fiber doped PPS (PPS65GF) were prepared by injection molding and provided by Shanghai Tongyao Medical Co., Ltd.

[0100] The pins were cleaned by ethanol, ultrasonic, or plasma to remove organic contaminants and particulate before metal-plating. The gold coating was done by vacuum magnetron and gold was only plated on the end face of one end of the cylindrical pin. The thickness of the gold layer was about 200nm controlled by the parameters of magnetron sputtering. All biological coating, immune response and signal detection were carried out on this gold surface.

[0101] Example 2. Protocols of IL6, LH, and FSH Assay

[0102] The assay format of interleukin 6 (IL6). luteinizing hormone (LH). and follicle-

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[0104] 183895916 1 stimulating hormone (FSH) is illustrated in FIG. 3.

[0105] Anti-IL6, anti-LH, and anti -FSH antibody coated pins were prepared according to the following coating steps. First, the gold-plated pins were cleaned with a pin cleaning reagent. Then, the cleaned pins were immersed in a microwell containing 220 pL of anti-fluorescein isothiocyanate (FITC) antibody conjugated to crosslinked FICOLL solution diluted in PBS buffer. Third, the pines were immersed in a microwell containing 220 pL fluorescein-labeled anti-IL6 antibody, fluorescein-labeled anti-LH antibody, or fluorescein-labeled anti-FSH antibody. Finally, the anti-IL6, anti-LH, and anti-FSH coated gold-plated pins were washed with PBST (PBS-Tween 20) and blocked with skim milk.

[0106] The pins coated with IL6, LH, or FSH captured antibody were used to perform fluorescent immunoassays in microplates in a fully automated instrument. The fully automated instrument includes an orbital mixer capable of holding, shaking, and moving the microplates, a pin holding mechanism, and a fluorescent reading system. The fluorescence reading system is shown in FIG. 1.

[0107] The assay format is illustrated in FIG. 3.

[0108] Each of the anit-IL6, anti-LH or anti-FSH antibody-coated pin tip was immersed in a microwell containing 200 pL IL-6, LH, or FSH samples diluted in assay buffer (PBS, 5 mg / ml BSA, 0.05% Tween 20). The micro wells were positioned on an orbital mixer (Big Bear Automation) with the pins held stationary, and the microwell were moved at 1200 rpm for 30 minutes. After the 30-minute incubation, each pin was washed 3 times with PBST (PBS. 0.05 % Tween 20). Then each pin was immersed in a microwell containing a biotinylated anti-IL6 antibody, a biotinylated anti-LH antibody, or a biotinylated anti-FSH antibody, and incubated for 1 minute at 1200 rpm. After 3 PBST washings, the fluorescence of the pin was read with a coaxial light optics as show n in FIG. 1 and recorded as pre-read.

[0109] Then the pin was immersed in a microwell containing Cy5-streptavidin-crosslinked FICOLL® at 5 pg / ml in assay buffer (containing 0.5% BSA and 0.02% mouse IgG in PBST). After 30 second incubation at 1200 rpm shaking, the pin was washed 4 times with PBST. Then the fluorescence of Cy5 bound to the pin w as measured, and record as the second fluorescence reading. The calculated fluorescent signal was the second fluorescence reading minus the pre-reading of pin. The calculated fluorescent signal was positively correlated with the antigen concentration.

[0110] - 13 -

[0111] 183895916 1 Example 3. Comparing IL6 Assay Performance on Gold-plated PPS vs. Gold-plated PPS doped with glass fiber

[0112] Gold-plated pins made of PPS, gold-plated pins made of PPS doped with 40% glass fibers, gold-plated pins made with PPS doped with 65% glass fibers, and gold-plated pins made of PA, all coated with anti-IL6 antibody, were prepared according to Examples 1 and 2. IL6 assays were performed according to Example 2. and the results are summarized in Table 1 (n=8 each). Table 1 shows the average (AVG), coefficient of variance (CV) of immunofluorescence signals and the signal-to-noise ratio (SNR) of gold-plated pins made with different materials. The results of Table 1 show that gold-plated PPS pins doped with glass fibers were better than gold-plated PPS pins without glass fiber, in terms of SNR and assay variance (CV).

[0113] Table 1. Performance Comparison

[0114] Example 4. Comparing Assay Performance of Present Assays with Commercial Assays

[0115] Gold-plated pins made of PPS doped with 65% glass fibers, coated with anti-LEI or anti-FSH antibody were prepared according to Examples 1 and 2. LH and FSH assays were performed according to Example 2, and the results are summarized in FIGs. 4 and 5.

[0116] The limit of detection (LOD) is the lowest concentration of a substance that can be consistently detected with a certain level of certainty. There are multiple ways to calculate the LOD, including. The LOD can be calculated by quantitating mean background signals plus 3.3 times standard deviation of background signal against a standard curve.

[0117] As shown in FIG. 4, the linear range of the present immunoassay of LH by using gold-plated PPS doped with 65% glass fiber pin was from 3.05 mIU to 250.000 mIU. The LOD was calculated as 3.05 mIU.

[0118] As shown in FIG. 5, the linear range of the present immunoassay of FSH by using gold-

[0119] - 14 -

[0120] 183895916 1 plated PPS doped with 65% glass fiber pin was from 1.67 mIU to 250,000 mIU. The LOD was calculated as 1.67 mIU.

[0121] We compare our LH and FSH test results with those from commercial test kits of Roche Elecsys LH and Elecsys FSH (instrument Cobas e 402; Cobas e 801), Beckman Access hLH and Access hFSH (Dxl Access Immunoassay Analyzers), Siemens Atellica IM Luteinizing Hormone (LH) and Atellica IM Follicle Stimulating Hormone (FSH) (Atellica IM Analyzer), and Abbott Elecsys LH and Elecsys FSH (Alinity / Architect). The linear range and LOD of each commercial kit are obtained from the kit instructions of the manufacturer.

[0122] The assay performance of our LH and FSH assays vs. commercial assays are summarized in Tables 2 and 3 below, which show that present assay provides better sensitivity than the commercial assays.

[0123] Table 2. LH Assay

[0124] Table 3. FSH Assay

[0125] The invention, and the manner and process of making and using it, are now described in such full, clear, concise and exact terms as to enable any person skilled in the art to which it pertains, to make and use the same. It is to be understood that the foregoing describes preferred embodiments of the present invention and that modifications may be made therein

[0126] - 15 -

[0127] 183895916 1 without departing from the scope of the present invention as set forth in the claims. To particularly point out and distinctly claim the subject matter regarded as invention, the following claims conclude this specification.

[0128] - 16 -

[0129] 183895916 1

Claims

WHAT IS CLAIMED IS:

1. A detection system for measuring a fluorescent signal in a fluorescent assay, comprising: a solid surface bound with a fluorescent label; a light source for emitting excitation light to a sensing surface of a solid surface; a light collecting lens pointed toward the sensing surface for collecting the emission fluorescent light; and an optical detector for detecting the emission fluorescent light; where the collecting lens collects and directs the emission fluorescent light to the optical detector; wherein the solid is made of plastic doped with glass fiber and the solid surface is plated with gold, and the hardness of the solid is at least 75 based on Rockwell M hardness scale.

2. The detection system of claim 1, wherein the plastic is polyphenylene sulfide (PPS), polyamide (PA), polycarbonate (PC), poly methyl methacry late (PMMA), polysty rene (PS), acry lonitrile butadiene sty rene (ABS), poly butylene terephthalate (PBT), poly etherimide (PEI), polyethersulfone (PESU). polymethylpentene(PMP). polyphthalamide (PPA). polyphenyl sulfone (PPSU), polysulfone (PSU), styrene methyl methacrylate(SMMA), thermoplastic polyimide (TPI), polyimide (PI), polyetheretherketone (PEEK), polyoxymethylene (POM), polyvinylidene fluoride (PVDF), polyethylene terephthalate (PET), liquid crystal polymer (LCP), cellulose acetate (CA), ethylene vinyl alcohol (EV OH), polyamide-imide (PAI), polyacrylonitrile (PAN), polyarylamide (PARA), styreneacrylonitrile copolymer (SAN), or any combination thereof.

3. The detection system of claim 2, wherein the plastic is PPS, PA, PC, PMMA, PS, or ABS.

4. The detection system of claim 2, wherein the solid is PPS doped with 30-70% glass fiber, PC doped with 20-40% glass fiber, PC doped with 20-40% glass fiber, ABS doped with 20-40% glass fiber, PA doped with 15-30% glass fiber, PARA doped with 30% -60% glass fiber, PBT doped with at least 30% glass fiber, PEEK doped with 20-40% glass fiber, PEI doped with 20-40% glass fiber, PPA doped with at least 45% glass fiber, PSU doped with at17183895916 1least 30% glass fiber. SAN doped with 20% glass fiber, or PS doped with 20-40% glass fiber.

5. The detection system of claim 4, wherein the solid is PPS doped with 65% or 40% glass fiber.

6. The detection system of claim 3, wherein the thickness of the gold coating on the solid surface is 100-500 nm.

7. The detection system of any one of claims 1-6. wherein the solid is in a form of a chip, a microwell, or a pin.

8. The detection system of claim 7, wherein the solid is a pin having an aspect ratio of length to width at least 5 to 1, the pin having a distal end and a proximal end. and the solid surface is a sensing surface at the proximal end.

9. The system according to claim 7, wherein the pin sensing surface has a diameter equal to or less than 5 mm.

10. The system according to any one of claims 1-9, wherein the excitation light is emitted perpendicular to the solid surface.18183895916 1

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